Synthetic linear apelin mimetics for the treatment of heart failure

ABSTRACT

The invention relates to novel compositions comprising modified apelin-13 peptide sequences designed to treat cardiovascular disease in subjects to whom they are administered, and which exhibit greater resistance to degradation, and equivalent or greater bioactivity than their wild type counterparts. The invention also relates to methods of making said compositions and using said compositions as pharmaceutically active agents to treat cardiovascular disease.

FIELD OF THE INVENTION

The invention relates to novel compositions comprising modified peptideand polypeptide sequences designed to treat cardiovascular disease insubjects to whom they are administered, and which exhibit greaterresistance to degradation, and equivalent or greater bioactivity thantheir wild type counterparts. The invention also relates to methods ofmaking said compositions and using said compositions as pharmaceuticallyactive agents to treat cardiovascular disease.

BACKGROUND OF THE INVENTION

The incidence of heart failure in the Western world is approximately1/100 adults after 65 yrs of age. The most common pathology is a chronicdeficit in cardiac contractility and, thereby, cardiac output, i.e., theeffective volume of blood expelled by either ventricle of the heart overtime. Patients with chronic heart failure can have acute episodes ofdecompensation, i.e., failure of the heart to maintain adequate bloodcirculation, where cardiac contractility declines further. There are˜500K hospitalizations per year for “acute decompensated heart failure”(ADHF) in the USA alone.

Current therapies for ADHF include diuretics, vasodilators, andinotropes, which directly increase cardiac contractility. Currentintravenous inotropes (dobutamine, dopamine, milrinone, levosimendan)are used in the acute setting, despite their association with adverseevents such as arrhythmia and increased long-term mortality. Theseliabilities have prevented their application in chronic heart failure.Digoxin is an oral inotrope, but is limited by a narrow therapeuticindex, increased arrhythmogenic potential and contraindication in renalinsufficiency.

A therapy for heart failure that increases cardiac contractility withoutarrhythmogenic or mortality liabilities is urgently needed for ADHF, butcould also address the enormous unmet medical need in chronic heartfailure.

Apelin is the endogenous ligand for the previously orphanG-protein-coupled receptor (GPCR), APJ, also referred to as apelinreceptor, angiotension-like-1 receptor, angiotension II-like-1 receptor,and the like. The apelin/APJ pathway is widely expressed in thecardiovascular system and apelin has shown major beneficialcardiovascular effects in preclinical models. Acute apelinadministration in humans causes peripheral and coronary vasodilatationand increases cardiac output (Circulation. 2010; 121:1818-1827). As aresult, APJ agonism is emerging as an important therapeutic target forpatients with heart failure. Activation of the apelin receptor APJ isthought to increase cardiac contractility and provide cardioprotection,without the liabilities of current therapies. However, the nativeapelins exhibit a very short half life and duration of action in vivo.The very short half life is a recognized major difficulty with thedelivery of such therapeutic endogenous peptides due to rapid serumclearance and proteolytic degradation via the action of peptidases.

One way which has been currently used to overcome this disadvantage isto administer large dosage of therapeutic peptide of interest to thepatient so that even if some therapeutic peptide is degraded, enoughremains to be therapeutically effective. However, this method isuncomfortable to patients. Since most therapeutic peptides cannot beadministered orally, the therapeutic peptide would have to be eitherconstantly infused, frequently infused by intravenous injection oradministered frequently by the inconvenient route of subcutaneousinjections. The need for frequent administration also results in manypotential peptide therapeutics having an unacceptable high projectedcost of treatment. The presence of large amounts of degraded peptide mayalso generate undesired side effects.

Discomfort in administration and high costs are ywo reasons why mosttherapeutic peptides with attractive bioactivity profiles may not bedeveloped as drug candidates.

Therefore, one approach to prolong half-life of peptides is to modifythe therapeutic peptides in such a way that their degradation is sloweddown while still maintaining biological activity.

It is therefore desirable to identify peptides and polypeptides thatmimic the function of apelin, but have increased half-life anddemonstrate equivalent or greater bioactivity than the naturallyoccurring apelin. Furthermore, it is desirable to identify apelin analogpeptides and polypeptides which exhibit increased conformationalconstraints, i.e., the ability to achieve and maintain an activeconformational state such that the peptides and polypeptides caninteract with their receptors and/or other pathway targets without theneed for additional folded or repositioning. Additional approachesincludes reducing the rate of clearance by conjugating the peptides tomolecules that prevent their elimination through kidney.

There is thus a need for modified therapeutic peptides with increasedhalf-life in order to provide longer duration of action in vivo, whilemaintaining low toxicity yet retaining the therapeutic advantages of themodified peptides.

SUMMARY OF THE INVENTION

This invention is directed to overcoming the problem of peptidedegradation in the body by modifying the therapeutic peptide orpolypeptide of interest, i.e. APJ agonists.

The aim of the present invention is to provide novel peptides andpolypeptides, or bioconjugates thereof, which are useful as APJagonists, and which also possess at least one of the followingimprovements over wild type apelin and other known apelin analogs:increased half-life; greater immunity to degradation upon administrationand/or upon solubilization; and increased conformational constraints,all while exhibiting the same or greater biological activity as wildtype apelin. The peptides and polypeptides of this invention, orbioconjugate thereof, are thus particularly useful for the treatment orprevention of cardiovascular diseases such as heart failure, disordersand conditions associated with heart failure, and disorders andconditions responsive to the activation of APJ receptor activity.

In one embodiment, the peptides and polypeptides of the invention, orbioconjugates thereof, are particularly useful for the treatment orprevention of a disorder or condition associated with heart failure, ora disorder responsive to the activation (or agonism) of the APJ receptoractivity. In another embodiment, the peptides and polypeptides of theinvention, or bioconjugates thereof, are useful in the treatment ofacute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

The invention pertains to the peptides and polypeptides, orbioconjugates thereof, pharmaceutical compositions, and methods ofmanufacture and use thereof, as described herein. Examples of peptidesand polypeptides of the invention include the peptides and polypeptidesaccording to any one of Formulae I to IV, or an amide, an ester, a saltthereof, and bioconjugate thereof, as well as any peptides orpolypeptides specifically listed herein, and bioconjugates thereof,including but not limited to the experimental examples.

The invention therefore provides a peptide or a polypeptide formula (I):

X1-X2-X3-R-X5-X6-X7-X8-X9-X10-X11-X12-X13  I

wherein:X1 is the N-terminus of the polypeptide and is absent, Q, A or pE;

X2 is R or r; X3 is P or 4-PhP; X5 is L, Cha, D-L, F, Y, Y(Bzl),3,4-Cl2-F or Nal;

X6 is a D-amino acid, S or A;X7 is a D-amino acid, L, H or Aib; and at least one of X6 and X7 isD-amino acid or Aib;

X8 is K, k, Q or E; X9 is G or D;

X10 is P or pipecolic acid;

X11 is D-Nle, Nle, f or D-Nva;

X12 is absent, P or a D-amino acid;X13 is the C-terminus and is absent, F or a D-amino acid; and at leastone of X11, X12 and X13 is a D-amino acid;wherein:

Nle is L-norleucine; D-Nle is D-norleucine; Nal is L-(naphthyl)alanine;D-Nva is D-norvaline;

Aib is α-aminoisobutyric acid;Cha is (S)-β-cyclohexylalanine;D-Tic is D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;pE is L-pyroglutamic acid;3,4-Cl2-F is (S)-3,4-dichlorophenylalanine;

Y is L-tyrosine; and Y(Bzl) is L-benzyl-tyrosine;

or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

As further explained herein, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D,” the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the L-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe D-amino acid.

Any of the above-listed amino acid residues of Formula I, or its relatedformulae described herein, e.g., Formulae I to IV, may be substituted ina conservative fashion, provided the peptide or polypeptide of theinvention still retains functional activity and structural properties(e.g., half-life extension, protection from degradation, conformationalconstraint). Principle and examples of permissible conservative aminoacid substitutions are further explained herein.

The invention further provides a bioconjugate or a multimer thereof,comprising:

-   -   a. a peptide or a polypeptide of anyone of Formulae I to IV,    -   b. a half-life extending moiety;    -   wherein said peptide or polypeptide and said half life are        covalently linked, optionally via a linker.

The half-life extending moiety of the invention can be covalentlyattached, linked or conjugated to a peptide or polypeptide analog. Ahalf-life extending moiety can be, for example, a polymer, such aspolyethylene glycol (PEG), a cholesterol group, a carbohydrate oroligosaccharide; or any natural or synthetic protein, polypeptide orpeptide that binds to a salvage receptor. Preferably, the half-lifeextending moiety is covalently linked, optionally via a linker, toplasma protein (albumin and immunoglobulin) with long serum half-lives.For example, the half-life extending moiety is an IgG constant domain orfragment thereof (e.g., the Fc region), Human Serum Albumin (HSA), oralbumin-binding polypeptides. Preferably, the half-life extending moietyportion of the bioconjugate is a human serum albumin.

The half-life extending moiety is attached in such a way so as enhance,and/or not to interfere with, the biological function of the constituentportions of the bio-conjugates of the invention, e.g., the peptide orpolypeptide of Formula I′, or its related formulae described herein(Formulae I-IV). The half-life extending moiety can be a protein such asan IgG constant domain or fragment thereof (e.g., the Fc region), HumanSerum Albumin (HSA), or albumin-binding polypeptides. Such proteinsdisclosed herein can also form multimers.

In some embodiments, the half-life extending moiety is covalently linkedto the N-terminus of the peptide or polypeptide of any one of FormulaeI-IV. In other embodiments, the half-life extending moiety is covalentlylinked to C-terminus of the peptide or polypeptide of any one ofFormulae I to IV of the invention.

The polypeptides of the invention or bioconjugate thereof, viaactivation of the APJ receptor, have utility in the treatment of acutedecompensated heart failure (ADHF), chronic heart failure, pulmonaryhypertension, atrial fibrillation, Brugada syndrome, ventriculartachycardia, atherosclerosis, hypertension, restenosis, ischemiccardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia,water retention, diabetes (including gestational diabetes), obesity,peripheral arterial disease, cerebrovascular accidents, transientischemic attacks, traumatic brain injuries, amyotrophic lateralsclerosis, burn injuries (including sunburn) and preeclampsia.

In a preferred embodiment the polypeptides of the invention, orbioconjugates thereof are useful in the treatment of acute decompensatedheart failure (ADHF).

In another embodiment, the invention pertains to a method for treatingdisorder or disease responsive to the activation of the APJ receptor, ina subject in need of such treatment, comprising: administering to thesubject an effective amount of a polypeptide according to any one ofFormulae I to IV, or an amide, an ester, a salt or a bioconjugatesthereof, such that the disorder or disease responsive to the activationof the APJ receptor in the subject is treated.

In yet another embodiment, the invention pertains to pharmaceuticalcompositions, comprising a polypeptide according to anyone of Formulae Ito IV, or an amide, an ester or salt thereof, or a bioconjugate thereof,and one or more pharmaceutically acceptable carriers.

In still another embodiment, the invention pertains to combinationsincluding, a polypeptide according to any one of Formulae I to IV, or anamide, an ester, a salt or a bioconjugate thereof, and pharmaceuticalcombinations of one or more therapeutically active agents.

In another embodiment, the invention pertains to a method for activationof the APJ receptor in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of apolypeptide according to anyone of Formulae I to IV, or an amide, anester, a salt or a bioconjugate thereof.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of interpreting this specification, the followingdefinitions will apply unless specified otherwise and wheneverappropriate, terms used in the singular will also include the plural andvice versa.

As used herein, “disorders or diseases responsive to the modulation ofthe APJ receptor,” “disorders and conditions responsive to themodulation of the APJ,” “disorders and conditions responsive to themodulation of APJ receptor activity,” “disorders responsive to theactivation (or agonism) of the APJ receptor activity,” and like termsinclude acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

As used herein, “Activation of APJ receptor activity,” or “Activation ofthe APJ receptor,” refers to an increase in the APJ receptor activity.The activation of the APJ receptor activity is also referred to as“agonism” of the APJ receptor, e.g., by administration of the peptidesand polypeptides of the invention.

As used herein, the terms “polypeptide” and “peptide” are usedinterchangeably to refer to two or more amino acids linked together.Except for the abbreviations for the uncommon or unnatural amino acidsset forth in Table 1 below, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D”, the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the D-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe L-amino acid. Groups or strings or amino acid abbreviations are usedto represent peptides. Peptides are indicated with the N-terminus on theleft and the sequence is written from the N-terminus to the C-terminus.

Peptides of the invention contain non-natural amino acids (i.e.,compounds that do not occur in nature) and other amino acid analogs asare known in the art may alternatively be employed.

Certain non-natural amino acids can be introduced by the technologydescribed in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wangand Schultz, Science 301:964-967, 2003; Wang et al., Science292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in U.S.Pat. No. 7,083,970. Briefly, some of these expression systems involvesite-directed mutagenesis to introduce a nonsense codon, such as anamber TAG, into the open reading frame encoding a polypeptide of theinvention. Such expression vectors are then introduced into a host thatcan utilize a tRNA specific for the introduced nonsense codon andcharged with the non-natural amino acid of choice. Particularnon-natural amino acids that are beneficial for purpose of conjugatingmoieties to the polypeptides of the invention include those withacetylene and azido side chains.

One or more of the natural or un-natural amino acids in a peptide of theinvention may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Saidmodifications may be done in a site-specific or non-site-specificmanner. In a preferred embodiment, the modifications of the peptide leadto a more stable peptide (e.g., one exhibiting greater half-life invivo). These modifications may include the incorporation of additionalD-amino acids, etc. None of the modifications should substantiallyinterfere with the desired biological activity of the peptide, but suchmodifications may confer desirable properties, e.g., enhanced biologicalactivity, on the peptide.

Said modifications enhance the biological properties of the proteins ofthe invention relative to the wild-type proteins, as well as, in somecases, serving as points of attachment for, e.g., labels and proteinhalf-life extension agents, and for purposes of affixing said variantsto the surface of a solid support.

In certain embodiments, such modifications, e.g., site-specificmodifications, are used to attach conjugates, e.g., PEG groups topolypeptides, and/or peptides of the invention, for purposes of, e.g.,extending half-life or otherwise improving the biological properties ofsaid polypeptides, and/or peptides. Said techniques are describedfurther herein.

In other embodiments, such modifications, e.g., site-specificmodifications, are used to attach other polymers and small molecules andrecombinant protein sequences that extend half-life of the polypeptideof the invention. One such embodiment includes the attachment of fattyacids or specific albumin binding compounds to polypeptides, and/orpeptides. In other embodiments, the modifications are made at aparticular amino acid type and may be attached at one or more sites onthe polypeptides.

In other embodiments, such modifications, e.g., site-specificmodifications, are used as means of attachment for the production ofwild-type and/or variant multimers, e.g., dimers (homodimers orheterodimers) or trimers or tetramers. These multimeric proteinmolecules may additionally have groups such as PEG, sugars, and/orPEG-cholesterol conjugates attached or be fused either amino-terminallyor carboxy-terminally to other proteins such as Fc, Human Serum Albumin(HSA), etc.

In other embodiments, such site-specific modifications are used toproduce proteins, polypeptides and/or peptides wherein the position ofthe site-specifically incorporated pyrrolysine or pyrrolysine analogueor non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe)allows for controlled orientation and attachment of such proteins,polypeptides and/or peptides onto a surface of a solid support or tohave groups such as PEG, sugars and/or PEG-cholesterol conjugatesattached.

In other embodiments, such site-specific modifications are used tosite-specifically cross-link proteins, polypeptides and/or peptidesthereby forming hetero-oligomers including, but not limited to,heterodimers and heterotrimers. In other embodiments, such site-specificmodifications are used to site-specifically cross-link proteins,polypeptides and/or peptides thereby forming protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates. In other embodiments, a site specificmodification may include a branching point to allow more than one typeof molecule to be attached at a single site of a protein, polypeptide orpeptide.

In other embodiments, the modifications listed herein can be done in anon-site-specific manner and result in protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates of the invention.

In some embodiments, the present invention provides complexes whichcomprise at least one peptide or polypeptide of anyone of Formulae I-IVbound to an antibody, such as an antibody why specifically binds apeptide or polypeptide as disclosed herein.

One of ordinary skill in the art will appreciate that various amino acidsubstitutions, e.g, conservative amino acid substitutions, may be madein the sequence of any of the polypeptides described herein, withoutnecessarily decreasing its activity. As used herein, “amino acidcommonly used as a substitute thereof” includes conservativesubstitutions (i.e., substitutions with amino acids of comparablechemical characteristics). For the purposes of conservativesubstitution, the non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophanand methionine. The polar (hydrophilic), neutral amino acids includeserine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of amino acid substitutions includesubstituting an L-amino acid for its corresponding D-amino acid,substituting cysteine for homocysteine or other non natural amino acidshaving a thiol-containing side chain, substituting a lysine forhomolysine, diaminobutyric acid, diaminopropionic acid, ornithine orother non natural amino acids having an amino containing side chain, orsubstituting an alanine for norvaline or the like.

The term “amino acid,” as used herein, refers to naturally occurringamino acids, unnatural amino acids, amino acid analogues and amino acidmimetics that function in a manner similar to the naturally occurringamino acids, all in their D and L stereoisomers if their structureallows such stereoisomeric forms. Amino acids are referred to herein byeither their name, their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “naturally occurring” refers to materials which are found innature and are not manipulated by man. Similarly, “non-naturallyoccurring,” “un-natural,” and the like, as used herein, refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man. When used in connection with aminoacids, the term “naturally occurring” refers to the 20 conventionalamino acids (i.e., alanine (A or Ala), cysteine (C or Cys), asparticacid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe),glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine(K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N orAsn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg),serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan(W or Trp), and tyrosine (Y or Tyr)).

The terms “non-natural amino acid” and “unnatural amino acid,” as usedherein, are interchangeably intended to represent amino acid structuresthat cannot be generated biosynthetically in any organism usingunmodified or modified genes from any organism, whether the same ordifferent. The terms refer to an amino acid residue that is not presentin the naturally occurring (wild-type) apelin protein sequence or thesequences of the present invention. These include, but are not limitedto, modified amino acids and/or amino acid analogues that are not one ofthe 20 naturally occurring amino acids, selenocysteine, pyrrolysine(Pyl), or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCTpatent publication WO2010/48582). Such non-natural amino acid residuescan be introduced by substitution of naturally occurring amino acids,and/or by insertion of non-natural amino acids into the naturallyoccurring (wild-type) Apelin protein sequence or the sequences of theinvention. The non-natural amino acid residue also can be incorporatedsuch that a desired functionality is imparted to the apelin molecule,for example, the ability to link a functional moiety (e.g., PEG). Whenused in connection with amino acids, the symbol “U” shall mean“non-natural amino acid” and “unnatural amino acid,” as used herein.

In addition, it is understood that such “unnatural amino acids” requirea modified tRNA and a modified tRNA synthetase (RS) for incorporationinto a protein. These “selected” orthogonal tRNA/RS pairs are generatedby a selection process as developed by Schultz et al. or by random ortargeted mutation. As way of example, pyrroline-carboxy-lysine is a“natural amino acid” as it is generated biosynthetically by genestransferred from one organism into the host cells and as it isincorporated into proteins by using natural tRNA and tRNA synthetasegenes, while p-aminophenylalanine (See, Generation of a bacterium with a21 amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang L,Martin A B, King D S, Horn D M, Schultz P G. J Am Chem Soc. 2003 January29; 125(4):935-9) is an “unnatural amino acid” because, althoughgenerated biosynthetically, it is incorporated into proteins by a“selected” orthogonal tRNA/tRNA synthetase pair.

Modified encoded amino acids include, but are not limited to,hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidinecarboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminoproprionic acid, N-ethylglycine, N-methylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.The term “amino acid” also includes naturally occurring amino acids thatare metabolites in certain organisms but are not encoded by the geneticcode for incorporation into proteins. Such amino acids include, but arenot limited to, ornithine, D-ornithine, and D-arginine.

The term “amino acid analogue,” as used herein, refers to compounds thathave the same basic chemical structure as a naturally occurring aminoacid, by way of example only, an α-carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group. Amino acid analoguesinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or their C-terminal carboxy group,their N-terminal amino group and/or their side-chain functional groupsare chemically modified. Such analogues include, but are not limited to,methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteinesulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alaninecarboxamide, homoserine, norleucine, and methionine methyl sulfonium.

TABLE 1 Un-natural or non-natural amino acids as described in theinvention: Symbol Name Structure Aib α-Aminoisobutyric acid

Nva or nva (D-Nva) L-Norvaline or D-Norvaline

1-Nal 1-Naphthalanine

2-Nal 2-Naphthalanine

Cha β-Cyclohexylalanine

Tic or tic (D-Tic) 1,2,3,4- Tetrahydroisoquinoline-3- carboxylic acid (Dor L)

Nle or nle (D-Nle) L-Norleucine or D-Norleucine

pE Pyroglutamic acid

4-PhP 4-Phenylproline

Pip Pipecolinic acid

Abu or abu (D-Abu) 2-amino-butyric acid

3,4-Cl2F (D or L) 3,4-dichlorophenylalanine

5-Aminovaleric acid

O2Oc 8-Amino-3,6-dioxaoctanoic acid

Nal refers to both 1-Naphthalanine and 2-Naphthalanine, preferably2-naphthalanine. 4-Phenylproline refers to both cis and trans4-phenylproline, preferably trans-4-phenylproline

As used herein the term “amide” refers to an amide derivative of thecarboxylic acid group at the C-terminus (e.g. —C(O)NH₂, —C(O)NH—C₁₋₆alkyl, —C(O)NH—C₁₋₂alkylphenyl, —C(O)NH—NHBn, —C(O)—4 phenoxypiperidineor —C(O)N(C₁₋₆ alkyl)₂).

The term “amide” also refers to derivatives of the amino group at theN-terminus (e.g. —NHC(O)C₁₋₁₆alkyl, —NHC(O)(CH₂)_(n)Ph (n is an integerof 1 to 6), —NHC(O)(CH₂)₂CO₂H, 4-Cl-Ph-(CH₂)₃C(O)NH—,C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—,C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—;C₁₅H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)NH—, Ph-CH₂CH₂NHC(O)—NH— orCH₃(OCH₂CH₂)_(m)C(O)NH— (m is an integer of 1 to 12).

As used herein, the term “ester” refers to an ester derivative of thecarboxylic acid group at the C-terminus (e.g —COOR) form wherein R ofthe ester refers to C₁₋₆ alkyl groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, etc., C₃₋₈ cycloalkyl groups such as cyclopentyl,cyclohexyl, etc., C₆₋₁₀ aryl groups such as phenyl, α-naphthyl, etc.,C₆₋₁₀ aryl-C₁₋₆ alkyl groups, for example phenyl-C₁₋₂ alkyl groups suchas benzyl, phenethyl, benzhydryl, etc., and α-naphthyl-C₁₋₂ alkyl groupssuch as α-naphthylmethyl and the like. Mention may also be made ofpivaloyloxymethyl ester and the like, which are commonly used as estersfor oral administration. When the polypeptides of the invention possessadditional carboxyl or carboxylate groups in positions other than the Cterminus, those polypeptides in which such groups are amidated oresterified also fall under the category of the polypeptide of theinvention. In such cases, the esters may for example be the same kindsof esters as the C-terminal esters mentioned above.

The term alkyl refers to a fully saturated branched or unbranched (orstraight chain or linear) hydrocarbon moiety, comprising 1 to 20 carbonatoms. Preferably the alkyl comprises 1 to 7 carbon atoms, and morepreferably 1 to 4 carbon atoms.

The term aryl refers to monocyclic or bicyclic aromatic hydrocarbongroups having 6-10 carbon atoms in the ring portion. Representativeexamples of aryl are phenyl or naphthyl.

The term heteroaryl includes monocyclic or bicyclic heteroaryl,containing from 5-10 ring members selected from carbon atoms and 1 to 5heteroatoms, and each heteroatoms is independently selected from O, N orS wherein S and N may be oxidized to various oxidation states. Forbicyclic heteroaryl system, the system is fully aromatic (i.e. all ringsare aromatic).

The term cycloalkyl refers to saturated or unsaturated but non-aromaticmonocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbonatoms, preferably 3-8, or 3-7 carbon atoms. For bicyclic, and tricycliccycloalkyl system, all rings are non-aromatic.

The term heterocyclyl refers to a saturated or unsaturated non-aromatic(partially unsaturated) ring which is a 4-, 5-, 6-, or 7-memberedmonocyclic, and contains at least one heteroatom selected from O, S andN, where the N and S can also optionally be oxidized to variousoxidation states. In one embodiment, heterocyclyl moiety represents asaturated monocyclic ring containing from 5-7 ring atoms and optionallycontaining a further heteroatom, selected from O, S or N.

The term “APJ” (also referred to as “apelin receptor,”“angiotension-like-1 receptor,” “angiotension II-like-1 receptor,” andthe like) indicates a 380 residue, 7 transmembrane domain, Gi coupledreceptor whose gene is localized on the long arm of chromosome 11 inhumans (NCBI Reference Sequence: NP_(—)005152.1, and encoded by NCBIReference Sequence: NM_(—)005161). APJ was first cloned in 1993 fromgenomic human DNA using degenerate oligonucleotide primers (O'Dowd etal. Gene, 136:355-60, 1993) and shares significant homology withangiotensin II receptor type 1. Despite this homology however,angiotensin II does not bind APJ. Although orphan for many years, theendogenous ligand has been isolated and named apelin (Tatemoto et al.,Biochem Biophys Res Commun 251, 471-6 (1998)).

The term “apelin,” indicates a 77 residue preprotein (NCBI ReferenceSequence: NP_(—)0059109.3, and encoded by NCBI Reference Sequence:NM_(—)017413.3), which gets processed into biologically active forms ofapelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13,apelin-12. The full length mature peptide, referred to as “apelin-36,”comprises 36 amino acids, but the most potent isoform is thepyroglutamated form of a 13mer of apelin (apelin-13), referred to as“Pyr-1-apelin-13 or Pyr¹-apelin-13” Different apelin forms aredescribed, for instance, in U.S. Pat. No. 6,492,324B1.

The term “conjugate” and “bioconjuagte” is used interchangeably and isintended to refer to the entity formed as a result of a covalentattachment between a polypeptide of anyone of Formulae I to IV, and ahalf-life extending moiety, optionally via linker.

The term half-life extending moiety can be covalently linked/attached toa peptide or polypeptide analog. A half-life extending moiety can be,for example, a polymer, such as polyethylene glycol (PEG), a cholesterolgroup, a carbohydrate or olisaccharide; or any natural or syntheticprotein, polypeptide or peptide that binds to a salvage receptor.Preferably, the half-life extending moiety is covalently linked,optionally via a linker, to plasma protein (albumin) with long serumhalf-lives. For example, the half-life extending moiety is Human SerumAlbumin (HSA), or albumin-binding polypeptides.

The term “increased half-life” or “increase serum half-life” or“extending half-life” is meant the positive change in circulatinghalf-life of a modified biologically active molecule (e.g. apelin 13)relative to its non-modified form (or naked form of the peptide). Serumhalf-life is measured by taking blood samples at various time pointsafter administration of the biologically active molecule, anddetermining the concentration of that molecule in each sample. Measuringthe change in serum concentration with time allows calculation of theserum half-life of a modified molecule (e.g. conjugated molecule). Bycomparing the serum half-life of a modified molecule (e.g. conjugatedmolecule), with an unmodified molecule (e.g. apelin 13), the relativeincrease in serum half-life or t½ may be determined. The increase isdesirably at least about two-fold, but a smaller increase may be useful.

Polypeptides of the Invention

Various embodiments of the invention are described herein. It will berecognized that features specified in each embodiment may be combinedwith other specified features to provide further embodiments.

In embodiment 1, the invention therefore provides a peptide or apolypeptide formula (I):

X1-X2-X3-R-X5-X6-X7-X8-X9-X10-X11-X12-X13  I

wherein:X1 is the N-terminus of the polypeptide and is absent, Q, A or pE;

X2 is R or r; X3 is P or 4-PhP; X5 is L, Cha, D-L, F, Y, Y(Bzl),3,4-Cl2-F or Nal;

X6 is a D-amino acid, S or A;X7 is a D-amino acid, L, H or Aib; and at least one of X6 and X7 isD-amino acid or Aib;

X8 is K, k, Q or E; X9 is G or D;

X10 is P or pipecolic acid;

X11 is D-Nle, Nle, f or D-Nva;

X12 is absent, P or a D-amino acid;X13 is the C-terminus and is absent, F or a D-amino acid; and at leastone of X11, X12 and X13 is a D-amino acid;wherein:

Nle is L-norleucine; D-Nle is D-norleucine; Nal is L-(naphthyl)alanine;D-Nva is D-norvaline;

Aib is α-aminoisobutyric acid;Cha is (S)-β-cyclohexylalanine;D-Tic is D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;pE is L-pyroglutamic acid;3,4-Cl2-F is (S)-3,4-dichlorophenylalanine;

Y is L-tyrosine; and Y(Bzl) is L-benzyl-tyrosine;

or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In embodiment 1a, the invention therefore provides a peptide or apolypeptide formula (IA):

X1-X2-X3-R-X5-X6-X7-X8-G-X10-X11-X12-X13  IA

wherein:X1 is the N-terminus of the polypeptide and is absent Q, A or pE;

X2 is R or r; X3 is P or 4-PhP; X5 is L, Cha, D-L, F, Y, Y(Bzl),3,4-Cl2-F or 2-Nal;

X6 is a D-amino acid or S;X7 is a D-amino acid, H or Aib; and at least one of X6 and X7 is D-aminoacid or Aib;

X8 is K or k;

X10 is P or pipecolic acid;

X11 is D-Nle or Nle;

X12 is absent, P or a D-amino acid;X13 is the C-terminus and is absent, F or a D-amino acid; and at leastone of X11, X12 and X13 is a D-amino acid;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In embodiment 1b, the invention pertains to a peptide or a polypeptideformula (I) or (IA) wherein X1 is either absent or pE, or an amide, anester or a salt of the polypeptide; or a polypeptide substantiallyequivalent thereto.

In one aspect of embodiment 1, 1A or 1B, the invention pertains topeptide or polypeptide of Formula I or IA wherein the amino group in theside chain of K or k is optionally linked to a fatty acid via an amidebond. In a further aspect of this embodiment, the fatty acid is selectedfrom lauroyl, myristoyl or palmitoyl, wherein lauroyl is C₁₁H₂₃C(O)—,myristoyl is C₁₃H₂₇C(O)— and palmitoyl is C₁₅H₃₁C(O)—.

In one aspect of embodiment 1, 1A or 1B, the invention pertains topeptide or polypeptide of Formula I or IA wherein the amino group in theside chain of K or k is optionally linked to a lipophilic group via anamide bond, wherein the lipophilic group is selected from a fatty acidas described supra and lauroyl(O2Oc), myristoyl(O2Oc) andpalmitoyl(O2Oc) and wherein lauroyl(O2Oc) isC₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—; myristoyl(O2Oc) isC₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—; palmitoyl(O2Oc) isC₁₅H₃₁C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—. Examples of side chain fattyacids have been described in U.S. provisional application No. 61/591,557(Attorney Docket Number PAT054961-US-PSP) filed on Jan. 27, 2012, whichis hereby incorporated by reference.

In embodiment 2, the invention pertains to a polypeptide according toembodiment 1, 1A or 1B, wherein X6 and X12 are D-amino acids; or anamide, an ester or a salt thereof.

In embodiment 3, the invention pertains to a polypeptide according toembodiment 2, wherein X13 is D-amino acid; or an amide, an ester or asalt thereof.

In embodiment 4, the invention pertains to a polypeptide according toembodiment 3, wherein X11 is D-amino acid; or an amide, an ester of asalt thereof.

In embodiment 5, the invention pertains to a polypeptide according toembodiment 1 1A or 1B, wherein X6 and X13 are D-amino acids; or anamide, an ester or a salt thereof.

In embodiment 6, the invention pertains to a polypeptide according toembodiment 1, 1A or 1B, wherein X7 and X12 are D-amino acids; or anamide, an ester or a salt thereof.

In embodiment 7, the invention pertains to a polypeptide according toembodiment 6 wherein X13 is a D-amino acid; or an amide, an ester or asalt thereof.

In embodiment 8, the invention pertains to a polypeptide accordinganyone of to embodiments 1 (1A or 1B) to 7 having the following formulaII:

X1-R-P-R-X5-a-X7-X8-G-P-X11-X12-X13  II

or an amide, an ester or a salt of the polypeptide.

In embodiment 9, the invention pertains to a polypeptide according toanyone of embodiments 1 (1A or 1B) to 8 having Formula III:

X1-R-P-R-X5-X6-X7-K-G-P-X11-a-f  III;

or an amide, an ester or a salt of the polypeptide.

In embodiment 10, the invention pertains to a polypeptide according toanyone of embodiments 1 (1A or 1B), 6, 7 and 9 having Formula IV:

X1-R-P-R-X5-S-X7-K-G-P-X11-X12-X13  IV;

or an amide, an ester or a salt of the polypeptide.

In embodiment 11, the invention pertains to a polypeptide according toanyone of embodiments 1 (1A or 1B) to 9, wherein X6 is a D-amino acidselected from a, D-Leu, k, s, d, nva, abu, f, h, v and D-Cys(tBu); or anamide, an ester or a salt thereof. In a further aspect of thisembodiment, X11 is nle or f.

In embodiment 12, the invention pertains to a polypeptide according toanyone of embodiments 1 (1A or 1B) to 11 wherein X7 is Aib or a D-aminoacid selected from a, f and h; or an amide, an ester or a salt thereof.

In embodiment 13, the invention pertains to a polypeptide according toanyone of embodiments 1 (1A or 1B) to 12 wherein X12 is absent or aD-amino acid selected from a, f, p, e, r, abu, nva, and D-Leu; or anamide, an ester or a salt thereof. In a further aspect of thisembodiment, X12 is a.

In embodiment 14, the invention pertains to a polypeptide according toanyone of embodiments 1 (1A or 1B) to 13 wherein X13 is absent or is aD-amino acid selected from f, y, d, and D-Tic, or an amide, an ester ora salt thereof. In a further aspect of this embodiment X13 is f.

In embodiment 15, the invention pertains to a polypeptide according toembodiment 14 wherein X13 is absent or f; or an amide, an ester or asalt of the polypeptide.

In embodiment 16, the invention pertains to a polypeptide according toanyone of the preceding embodiments wherein X1 is pE; or an amide, anester or a salt of the polypeptide.

In embodiment 16A, the invention pertains to a polypeptide according toanyone of the preceding embodiments wherein X1 is absent, A or Q; or anamide, an ester or a salt of the polypeptide. In a particular aspect ofthis embodiment, the peptide is covalently linked to a half-lifeextending moiety via its A or Q N-terminus

In embodiment 17, the invention pertains to a polypeptide according toanyone of embodiments 1 to 16 wherein X5 is L; or an amide, an ester ora salt of the polypeptide.

In embodiment 18, the invention pertains to a polypeptide according toanyone of embodiments 1 to 17 wherein X8 is K; or an amide, an ester ora salt of the polypeptide.

In embodiment 19, the invention pertains to a polypeptide according toanyone of embodiments 1 to 18 wherein X11 is Nle or nle; or an amide, anester or a salt of the polypeptide.

In embodiment 20, the invention pertains to a polypeptide according toanyone of the preceding embodiments wherein the C-terminus is an amide;or a salt of the polypeptide.

In embodiment 21, the invention pertains to a polypeptide according toembodiment 20 wherein the C-terminus is an amide of formula —C(O)—R2 andR2 is —NH₂, —NH—(CH₂)₂-Ph or 4-phenoxypiperidine; or a salt of thepolypeptide.

In another embodiment, the invention pertains to peptides andpolypeptides according to any one of Formulae I, IA, II, III or IV, orany of any other classes and subclasses described supra, (i.e. accordingto anyone of the embodiments 1 to 15 and 17-21) or an amide, an ester ora salt thereof, wherein X1 is absent; or an amide, an ester or a salt ofthe polypeptide. In one aspect of this embodiment, the N-terminus of thepeptide is an amide. In a further aspect of this embodiment, theinvention pertains to peptides and polypeptide according to any one ofFormulae I, IA, II, III or IV, or any of any other classes andsubclasses described supra, or an amide, an ester or a salt thereof,wherein the X1 is absent and the N-terminus is an amide of formula —NHRand R is CH₃C(O)—, CH₃—(O—CH₂CH₂)_(m)—C(O)—, palmitoyl(O2Oc)_(p),myristoyl(O2Oc)_(p), lauroyl(O2Oc)_(p) or Ph-CH₂CH₂NHC(O)—, benzoyl,phenacyl, succinyl, octanoyl, 4-phenylbutanoyl, 4-Cl-Ph-(CH2)₃C(O)—, orPh-CH₂CH₂NHC(O)—; and wherein

p is an integer 1 to 4;m is an integer 1 to 12;Lauroyl(O2Oc)_(p) is C₁₁H₂₃C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—;Myristoyl(O2Oc)_(p) is C₁₃H₂₇C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—;Palmitoyl(O2Oc)_(p) is C₁₅H₃₁C(O)[NH—(CH₂)₂—O—(CH²)²—O—CH²—C(O)]_(p)—.In one particular aspect of this embodiment R is acetyl, benzoyl,phenacyl, succinyl, octanoyl, 4-phenylbutanoyl, 4-Cl-Ph-(CH2)3C(O)—, orPh-CH₂CH₂NHC(O)—. Examples of N-terminus amides have been described inU.S. provisional application No. 61/591,557 (Attorney Docket NumberPAT054961-US-PSP) filed on Jan. 27, 2012, which is hereby incorporatedby reference.

In another embodiment, the invention pertains to peptides orpolypeptides according to anyone of Formulae I, IA, II, III or IV, or toany other classes and subclasses described supra, (i.e. according toanyone of embodiments 1 to 21), or an amide, an ester or a salt thereof,wherein N-terminus is an amide of formula NHR1 wherein R1 is CH₃C(O)—,CH₃—(O—CH₂CH₂)_(m)—C(O)—, palmitoyl(O2Oc), myristoyl(O2Oc),lauroyl(O2Oc) or Ph-CH₂CH₂NHC(O)—; and wherein m, lauroyl(O2Oc),myristoyl(O2Oc) and palmitoyl(O2Oc) are defined supra.

In another embodiment, the invention pertains to peptides andpolypeptides according to any one of Formulae I, IA, II, III or IV, orany of any other classes and subclasses described supra, (i.e. accordingto anyone of embodiments 1 to 8 and 10 to 21), wherein X13 is absent; oran amide, an ester or a salt thereof. In a particular aspect of thisembodiment, the C-terminus is an amide. In a further aspect of thisembodiment, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I, IA, II, III or IV, or any of anyother classes and subclasses described supra, or an amide, an ester or asalt thereof, wherein the C-terminus is an amide of formula —C(O)R2 andR2 is —NH₂, —NH-Me, —NH—NHBn, 4-Phenoxypiperidin-1-yl or —NH—(CH₂)₂-Ph.In a preferred aspect of this embodiment, the invention pertains topeptides and polypeptide according to any one of Formulae I to IV, orany of any other classes and subclasses described supra, or an amide, anester or a salt thereof, wherein the C-terminus is an amide of formula—C(O)R2 and R2 is —NH₂, —NH—(CH₂)₂-Ph or 4-phenoxypiperidin-1-yl.

In one embodiment, the invention pertains to a peptide or polypeptide ofanyone of embodiments 1 to 21, wherein three of the amino acids X1 toX13 are different from the corresponding amino acids present inPyr-1-apelin-13. In another embodiment, the invention pertains to apeptide or polypeptide of anyone of embodiments 1 to 21 wherein four ofthe amino acids X1 to X13 are different from the corresponding aminoacids present in Pyr-1-apelin-13.

In another embodiment, X1, X2, X3, X5, X6, X7, X8. X9, X10, X11, X12 andX13 amino acids are those defined by X1, X2, X3, X5, X6, X7, X8. X9,X10, X11, X12 and X13 amino acids in the Examples section below.

In another embodiment, individual polypeptides according to theinvention are those listed in the Examples section below or apharmaceutically acceptable salt thereof.

Unless specified otherwise, the term “polypeptide of the presentinvention” refers to a polypeptide of Formula (I) and subformulaethereof (Formulae IA, II, III or IV); or an amide, an ester or a saltthereof.

Unless specified otherwise, the terms “polypeptides of the presentinvention,” “peptides of the present invention,” “apelin peptideagonists,” and the like refer to peptides and polypeptides of Formula Iand subformulae thereof (Formulae IA, II, III or IV); or an amide, anester or a salt thereof. The peptides and polypeptides of the inventiondemonstrate substantially equivalent or improved activity and/or plasmastability over known apelin peptides and polypeptides described herein,including but not limited to wild type apelin, apelin-13 andpyr-1-apelin-13.

The peptides and polypeptides of the invention also encompass peptidesand polypeptides that are at least about 95% identical to the peptidesand polypeptides according to any one of Formulae I, IA, II, III or IV,or an amide, an ester or a salt thereof, as well as to any peptides orpolypeptides specifically listed herein, including but not limited tothe experimental examples.

As used herein, the phrase “homologous amino acid sequence,” orvariations thereof, refers to sequences characterized by a homology, atthe amino acid level, of at least a specified percentage and is usedinterchangeably with “sequence identity.” Homologous amino acidsequences include those amino acid sequences which contain conservativeamino acid substitutions and which polypeptides have the same bindingand/or activity. In some embodiments, an amino acid sequence ishomologous if it has at least 60% or greater, up to 99%, identity with acomparator sequence. In some embodiments, an amino acid sequence ishomologous if it shares one or more, up to 60, amino acid substitutions,additions, or deletions with a comparator sequence. In some embodiments,the homologous amino acid sequences have no more than 5 or no more than3 conservative amino acid substitutions.

Homology may also be at the polypeptide level. The degree or percentageidentity of peptides or polypeptides of the invention, or portionsthereof, and different amino acid sequences is calculated as the numberof exact matches in an alignment of the two sequences divided by thelength of the “invention sequence” or the “foreign sequence”, whicheveris shortest. The result is expressed as percent identity.

A polypeptide comprising an amino acid sequence having a homology ofabout 80-99.9%, preferably 90-99.9% to the amino acid sequence describedin the specific examples, and possessing a plasma stability superior toapelin-13 or pyr-1-apelin-13, fall under the category of the polypeptideof the invention. In one embodiment, the plasma stability improvement isat least 2 fold. In one embodiment, the polypeptide of the invention hasa plasma stability of at least 30 minutes. In another embodiment, thepolypeptide of the invention has a plasma stability of at least 60minutes, or at least 80 minutes, preferably at least 100 min and morepreferably at least 150 minutes.

The term “substantially equivalent” means the nature of thereceptor-binding activity, signal transduction activity and the like isequivalent. Thus, it is allowable that even differences among gradessuch as the strength of receptor binding activity and the molecularweight of the polypeptide are present.

A polypeptide as described herein, or a substantial equivalent thereto,by substitution, deletion, addition or insertion of one or more of aminoacids may be mentioned as polypeptides containing an amino acid sequencesubstantial equivalent(s) in the above sense. A polypeptide as describedherein, or a substantial equivalent thereto, by substitution of 1 to 5,preferably 1 to 3 and more preferably 1 or 2 amino acids with natural orun-natural amino acids may be mentioned as polypeptides containing anamino acid sequence substantial equivalent(s) in the above sense.Further modifications and alterations may include the replacement of anL-amino-acid with a D-amino acid, or other variation including, but notlimited to, phosphorylation, carboxylation, alkylation and the like aslong as the APJ agonistic activity of the peptide or polypeptide offormulae I, IA, II, III or IV is maintained and the plasma stability isimproved over the pyroglutamated form of apelin-13. For example, D-aminoacid are well tolerated with respect to activity and stability of thepolypeptide at position 2 (X2), position 6 (X6), position 7 (X7),position 8 (X8), position 11 (X11), position 12 (X12) and position 13(X13) of the linear peptides and polypeptides of Formulae I, IA, II, IIIor IV.

In embodiment 22, the invention further pertains to a bioconjugate or amultimer thereof, comprising:

-   -   a. a peptide or polypeptide of Formulae I, IA, II, III or IV, an        amide, salt or ester thereof, according to anyone of the        preceding embodiments;    -   b. a half-life extending moiety;        wherein said peptide or polypeptide and said half-life extending        moiety are covalently linked, optionally via a linker.

In embodiment 22A, the half-life extending moiety is covalently linkedto the N-terminus of the peptide of Formula I, IA, II, III or IV,optionally via a linker moiety.

In embodiment 22B, the half-life extending moiety is covalently linkedto the C-terminus of the peptide of Formula I, IA, II, III or IV,optionally via a linker moiety.

In embodiment 22C, the half-life extending moiety is covalently linkedto a side chain of the peptide of Formula I, IA, II, III or IV, e.g. thehalf-life is attached to an amino group in the side chain of K, Orn,Dab, Dap, hK or 4-amino-Isn, optionally via a linker moiety. Preferably,the half-life extending moiety is attached to the N-terminus of thepeptide of Formula I, IA, II, III or IV, optionally via a linker moiety.

In embodiment 23, the invention pertains to the bioconjugate or amultimer thereof, according to embodiment 19, wherein the half-lifeextending moiety is a human Serum Albumin.

In embodiment 24, the invention pertains to the bioconjugate accordingto embodiment 23 wherein the Human Serum Albumin is chemically linked tothe N-terminus of a polypeptide of any one of Formulae I to IV via alinker of the following Formulae:

wherein x is 1-20, R is linear or branched alkylene, cycloalkyl, aryl ofheteroaryl or combination thereof, R′ is linear or branched alkylene,aryl or cycloalkyl or combination thereof.

In embodiment 25, the invention pertains to the bioconjugate accordingto embodiment 22 or 23 wherein the Human Serum Albumin is chemicallylinked to the C-terminus of a polypeptide of any one of Formulae I to IVvia a linker of the following Formulae:

wherein x is 1-20, R is linear or branched alkylene, cycloalkyl, aryl ofheteroaryl or combination thereof, R′ is linear or branched alkylene,aryl or cycloalkyl or combination thereof.

Half-Life Extending Moiety

The half-life extending moiety of the invention can be covalentlyattached, linked or conjugated to a peptide or polypeptide analog. Ahalf-life extending moiety can be, for example, a polymer, such aspolyethylene glycol (PEG), a cholesterol group, a carbohydrate orolisaccharide; or any natural or synthetic protein, polypeptide orpeptide that binds to a salvage receptor. Preferably, the half-lifeextending moiety is covalently linked, optionally via a linker, toplasma protein (albumin and immunoglobulin) with long serum half-lives.For example, the half-life extending moiety is an IgG constant domain orfragment thereof (e.g., the Fc region), Human Serum Albumin (HSA), oralbumin-binding polypeptides. Preferably, the half-life extending moietyportion of the bioconjugate is human serum albumin.

Half-life extending moieties include Albumin, which refers to the mostabundant protein in the blood plasma having a molecular weight ofapproximately between 65 and 67 kilodaltons in its monomeric form,depending on species of origin. The term “albumin” is usedinterchangeably with “serum albumin” and is not meant to define thesource of albumin which forms a conjugate with the modified peptides ofthe invention. Thus, the term “albumin” as used herein may refer eitherto albumin purified from a natural source such as blood or serous fluis,or it may refer to chemically synthesized or recombinantly producedalbumin. Modified peptides or polypeptides of the invention arepreferentially tethered to the free thiol group of the cysteine-34 onthe surface of the albumin, optionally via a linker.

Half-life extending moieties include “native Fc” which refers tomolecule or sequence comprising the sequence of a non-antigen-bindingfragment resulting from digestion of whole antibody or produced by othermeans, whether in monomeric or multimeric form, and can contain thehinge region. The original immunoglobulin source of the native Fc ispreferably of human origin and can be any of the immunoglobulins,although IgG1 and IgG2 are preferred. Native Fc molecules are made up ofmonomeric polypeptides that can be linked into dimeric or multimericforms by covalent (i.e., disulfide bonds) and non-covalent association.The number of intermolecular disulfide bonds between monomeric subunitsof native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG,IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). Oneexample of a native Fc is a disulfide-bonded dimer resulting from papaindigestion of an IgG (see Ellison et al., 1982, Nucleic Acids Res. 10:4071-9). The term “native Fc” as used herein is generic to themonomeric, dimeric, and multimeric forms.

Half-life extending moieties include “Fc variant” which refers to amolecule or sequence that is modified from a native Fc but stillcomprises a binding site for the salvage receptor, FcRn (neonatal Fcreceptor). International Publication Nos. WO 97/34631 and WO 96/32478describe exemplary Fc variants, as well as interaction with the salvagereceptor, and are hereby incorporated by reference. Thus, the term “Fcvariant” can comprise a molecule or sequence that is humanized from anon-human native Fc. Furthermore, a native Fc comprises regions that canbe removed because they provide structural features or biologicalactivity that are not required for the bioconjugate of the invention.Thus, the term “Fc variant” comprises a molecule or sequence that lacksone or more native Fc sites or residues, or in which one or more Fcsites or residues has be modified, that affect or are involved in: (1)disulfide bond formation, (2) incompatibility with a selected host cell,(3) N-terminal heterogeneity upon expression in a selected host cell,(4) glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

Half-time extending moieties refer to “Fc domain” which encompassesnative Fc and Fc variants and sequences as defined above. As with Fcvariants and native Fc molecules, the term “Fc domain” includesmolecules in monomeric or multimeric form, whether digested from wholeantibody or produced by other means. In some embodiments of the presentinvention, an Fc domain can be conjugated to a polypeptide of Formula I′or anyone of Formulae I-IV via, for example, a covalent bond between theFc domain and the peptide sequence. Such Fc proteins can form multimersvia the association of the Fc domains and both these Fc proteins andtheir multimers are an aspect of the present invention.

Half-life extending moieties include “modified Fc fragment”, which shallmean an Fc fragment of an antibody comprising a modified sequence. TheFc fragment is a portion of an antibody comprising the CH2, CH3 and partof the hinge region. The modified Fc fragment can be derived from, forexample, IgGI, IgG2, IgG3, or IgG4.

The term “multimer” as applied to Fc domains or molecule comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently.

Linker

Any linker group is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer.

The linker is a chemical moiety that contains two reactivegroups/functional groups, one of which can react with the polypeptideand the other with the half-life extending moiety. The two reactivegroups of the linker are linked via a linking group, structure of whichis not critical as long as it does not interfere with the coupling ofthe linker to the peptide and the half-extending moiety.

The linker can be made up of amino acids linked together by peptidebonds. In some embodiments of the present invention, the linker is madeup of from 1 to 20 amino acids linked by peptide bonds, wherein theamino acids are selected from the 20 naturally occurring amino acids. Invarious embodiments, the 1 to 20 amino acids are selected from the aminoacids glycine, serine, alanine, proline, asparagine, glutamine, cysteineand lysine. In some embodiments, a linker is made up of a majority ofamino acids that are sterically unhindered, such as glycine and alanine.In some embodiments, linkers are polyglycines, polyalanines,combinations of glycine and alanine (such as poly(Gly-Ala)), orcombinations of glycine and serine (such as poly(Gly-Ser)). In otherembodiments, the linker comprises 1 to 20 amino acids which are selectedfrom unnatural amino acids. While a linker of 3-15 amino acid residuesis preferred for conjugation with the half-life extending moiety, thepresent invention contemplates linkers of any length or composition. Apreferred amino acid linker is O2Oc of the following formula:

or its repeating units.

The linkers described herein are exemplary, and linkers that are muchlonger and which include other residues are contemplated by the presentinvention. Non-peptide linkers are also contemplated by the presentinvention.

The linking portion of the linker may comprise one or more alkyl groups,alkoxy groups, alkenyl groups, cycloalkyl groups, aryl groups,heteroaryl groups and heterocyclic groups or combination thereof. Forexample, alkyl linkers such as such as —NH—(CH₂)z-C(O)— or—S—(CH₂)z-C(O)— or —O—(CH₂)z-C(O)— wherein z is 2-20 can be used. Thesealkyl linkers can further be substituted by any non-sterically hinderinggroup, including, but not limited to, a lower alkyl (e.g., C1-C6), loweracyl, halogen (e.g., Cl, Br), CN, NH2, or phenyl.

The linker can also be of polymeric nature. The linker may includepolymer chains or units that are biostable or biodegradable. Polymerswith repeat linkage may have varying degrees of stability underphysiological conditions depending on bond lability. Polymers maycontain bonds such as polycarbonates (—O—C(O)—O—), polyesters(—C(O)—O—), polyurethanes (—NH—C(O)—O—), polyamide (—C(O)—NH—). Thesebonds are provided by way of examples, and are not intended to limit thetype of bonds employable in the polymer chains or linkers of theinvention. Suitable polymers include, for example, polyethylene glycol(PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids,divinylether maleic anhydride, N-(2-hydroxypropyl)-methacrylicamide,dextran, dextran derivatives, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, starch and starch derivatives, polyalkyleneglycol and derivatives thereof, copolymers of polyalkylene glycols andderivatives thereof, polyvinyl ethyl ether, and the klike and mixturesthereof. A polymer linker is for example PEG. An exemplary non-peptidelinker is a polyethylene glycol linker:

wherein the linker has a molecular weight of 100 to 5000 kD, forexample, 100 to 500 kD.

Preferably, the linking moiety contains one or more amino acid moietiessuch as for example (O2Oc) unit or C₁₋₄alkylene-C(O)—, C₁₋₄alkylene,—NH—C₂₋₆alkylene-NH- or —NH—CH₂CH₂—O—CH₂CH₂—NH— diamino units orcombination thereof and the linking moiety links 2 reactive groups orfunctional groups.

Preferably, the reactive groups or functional groups are maleimide,thiol or pyridine-2-yldisulfanyl.

Preparation of Peptide or Polypeptide and Peptide-Linker Construct forAttachment to a Half-Life Extending Moiety:

The peptides and polypeptides of the present invention can be producedby the per se known procedures for peptide synthesis. The methods forpeptide synthesis may be any of a solid-phase synthesis and aliquid-phase synthesis. Thus, the peptide and polypeptide of interestcan be produced by condensing a partial peptide or amino acid capable ofconstituting the protein with the residual part thereof and, when theproduct has a protective group, the protective group is detachedwhereupon a desired peptide can be manufactured. The known methods forcondensation and deprotection include the procedures described in thefollowing literature (1)-(5).

-   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, 1966,-   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,    1965,-   (3) Nobuo Izumiya et al. Fundamentals and Experiments in Peptide    Synthesis, Maruzen, 1975,-   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment    Series 1, Protein Chemistry IV, 205, 1977, and-   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,    Peptide Synthesis, Hirokawa Shoten.

After the reaction, the peptide can be purified and isolated by acombination of conventional purification techniques such as solventextraction, column chromatography, liquid chromatography, andrecrystallization. Where the peptide isolated as above is a freecompound, it can be converted to a suitable salt by the known method.Conversely where the isolated product is a salt, it can be converted tothe free peptide by the known method.

The amide of polypeptide can be obtained by using a resin for peptidesynthesis which is suited for amidation. The resin includes chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenz-hydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, 2-chlorotritylchloride resin, and so on. Using such a resin, amino acids whose α-aminogroups and functional groups of side-chain have been suitably protectedare condensed on the resin according to the sequence of the objectivepeptide by various condensation techniques which are known per se. Atthe end of the series of reactions, the peptide or the protected peptideis removed from the resin and the protective groups are removed and ifnecessary, disulfide bonds are formed to obtain the objectivepolypeptide.

For the condensation of the above-mentioned protected amino acids, avariety of activating reagents for peptide synthesis can be used such asHATU, HCTU or e.g. a carbodiimide. The carbodiimide includes DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation with sucha reagent, a racemization inhibitor additive, e.g. HOBt or Oxyma Purecan be used. The protected amino acid can be directly added to the resinalong with the activation reagents and racemization inhibitor or bepre-activated as symmetric acid anhydride, HOBt ester, or HOOBt esterthen added to the resin. The solvent for the activation of protectedamino acids or condensation with the resin can be properly selected fromamong those solvents which are known to be useful for peptidecondensation reactions. For example, N,N-dimethylformamide,N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide,DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran,acetonitrile, ethyl acetate, or suitable mixtures of them can bementioned.

The reaction temperature can be selected from the range hitherto-knownto be useful for peptide bond formation and is usually selected from therange of about −20° C.-50° C. The activated amino acid derivative isgenerally used in a proportion of 1.5-4 fold excess. If the condensationis found to be insufficient by a test utilizing the ninhydrin reaction,the condensation reaction can be repeated to achieve a sufficientcondensation without removing the protective group. If repeatedcondensation still fails to provide a sufficient degree of condensation,the unreacted amino group can be acetylated with acetic anhydride oracetylimidazole.

The protecting group of amino group for the starting material amino acidincludes Z, Boc, tertiary-amyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, or Fmoc. The carboxy-protecting group that canbe used includes but is not limited to the above-mentioned C₁₋₆ alkyl,C₃₋₈ cycloalkyl and C₆₋₁₀aryl-C₁₋₂alkyl as well as 2-adamantyl,4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl,benzyloxycarbonylhydrazido, tertiary-butoxycarbonylhydrazido, andtritylhydrazido.

The hydroxy group of serine and threonine can be protected byesterification or etherification. The group suited for saidesterification includes carbon-derived groups such as lower alkanoylgroups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc.,benzyloxycarbonyl, and ethoxycarbonyl. The group suited for saidetherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.The protective group for the phenolic hydroxyl group of tyrosineincludes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, and tertiary-butyl.

The protecting group of imidazole for histidine includes Tos,4-methoxy-2,3,6-tri ethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes thecorresponding acid anhydride, azide and active esters, e.g. esters withalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc. The activatedamino group of the starting amino acid includes the correspondingphosphoramide.

The method for elimination of protective groups includes catalyticreduction using hydrogen gas in the presence of a catalyst such aspalladium black or palladium-on-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid, or a mixture of such acids, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, reductionwith sodium metal in liquid ammonia. The elimination reaction by theabove-mentioned acid treatment is generally carried out at a temperatureof −20° C.-40° C. and can be conducted advantageously with addition of acation acceptor such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. The2,4-dinitrophenyl group used for protecting the imidazole group ofhistidine can be eliminated by treatment with thiophenol, while theformyl group used for protecting the indole group of tryptophan can beeliminated by alkali treatment with dilute sodium hydroxide solution ordilute aqueous ammonia as well as the above-mentioned acid treatment inthe presence of 1,2-ethanedithiol, 1,4-butanedithiol.

The method for protecting functional groups which should not take partin the reaction of the starting material, the protective groups that canbe used, the method of removing the protective groups, and the method ofactivating the functional groups that are to take part in the reactioncan all be selected judicially from among the known groups and methods.

An another method for obtaining the amide form of the polypeptidecomprises amidating the -carboxyl group of the C-terminal amino acid atfirst, then extending the peptide chain to the N-side until the desiredchain length, and then selectively deprotecting the α-amino group of theC-terminal peptide and the α-carboxy group of the amino acid or peptidethat is to form the remainder of the objective polypeptide andcondensing the two fragments whose α-amino group and side-chainfunctional groups have been protected with suitable protective groupsmentioned above in a mixed solvent such as that mentioned hereinbefore.The parameters of this condensation reaction can be the same asdescribed hereinbefore. From the protected peptide obtained bycondensation, all the protective groups are removed by theabove-described method to thereby provide the desired crude peptide.This crude peptide can be purified by known purification procedures andthe main fraction be lyophilized to provide the objective amidatedpolypeptide. To obtain an ester of the polypeptide, the a-carboxyl groupof the C-terminal amino acid is condensed with a desired alcohol to givean amino acid ester and then, the procedure described above forproduction of the amide is followed.

The modified therapeutic peptides or polypeptides and/or peptide-linkerconstruct include reactive groups which can react with availablereactive functionalities on the half-life extending moiety to form acovalent bond. Reactive groups are chemical groups capable of forming acovalent bond. Reactive groups can generally be carboxy, phosphoryl,acyl group, ester or mixed anhydride, maleimide, imidate,pyridine-2-yl-disulfanyl, thereby capable of forming a covalent bondwith functionalities like amino group, hydroxyl group, carboxy group ora thiol group at the target site of the half-life extending moiety or ata chemically modified Fc domain as disclosed below. Reactive groups ofparticular interest for linking to an Albumin includemaleimido-containing groups and pyridine-2-yl-disulfanyl containinggroup.

Functionalities are groups the half life extending moiety to whichreactive groups on modified peptides or polypeptides are capable ofreacting with to form covalent bonds. Functionalities include hydroxylgroups for bonding with ester reactive entities, thiol groups forreacting with maleimides, maleimido-containing groups orpyridine-2-yldisulfanyl, imidates and thioester groups; and amino groupsfor bonding to carboxylic acid, phosphoryl groups, acyl groups.

Schemes 1 to 3 describe the synthesis of peptide-Linker constructwherein the peptide is a peptide according to anyone of Formulae I toIV.Scheme 1 describes the synthesis of a maleimide containing linkerattached to the N-terminus of a polypeptide of Formula I to IV.

The N-terminus of the peptide is coupled with one or more O2Oc aminoacid units (x is 1 to 20, preferably 1 to 10 and more preferably 3 to 6)according to well established amide coupling chemistry to generate (1A).The terminal amino functionality of (1A) is reacted with an activatedacid (1B) wherein R is linear or branched alkylene, aryl, heteroaryl,cycloalkyl or combination thereof, in order to generate thepeptide-maleimide containing linker construct (1C). The activated acid(1B) is commercially available or readily available from itscorresponding carboxylic acid according to technique known to someone ofordinary skill in the art. Preferably, R is a linear alkylene, and morepreferably R is —CH₂—CH₂—. Alternatively, for peptides containing anamino functionality in the side chain (for example peptide containing alysine), orthogonal protecting group such as Alloc is required prior tothe coupling reaction, followed by additional deprotection step in orderto obtain (1C).Scheme 2A and 2B describe the synthesis of pyridine-2-yl-disulfanylcontaining linker attached to the N-terminus of a polypeptide accordingto any one of Formula I to IV.

Peptide-Linker construct (1A) is prepared as described in Scheme 1 andis further reacted with an activated acid of Formula (2A) wherein R′ isa linear or branched alkylene, to generate apeptide-pyridine-2-yl-disulfanyl containing linker construct (2B).Activated acid (2A) is commercially available or is readily availablefrom its corresponding carboxylic acid according to techniques known tosomeone of ordinary skill in the art. Preferably R′ is —CH₂—CH₂—.Alternatively, Peptide-linker construct (2C) can be prepared usingHO₂C—R′—SH, or a protected form thereof (e.g. trityl or Acm groups,requiring additional deprotection steps), and further reacted with (2D)to generate peptide-pyridine-2-yl-disulfanyl containing linker construct(2B).Similar reactive groups are attached to the C-terminus of the peptide ina similar way as described in Schemes 1, 2A and 2B using a diamino unitsuch as for example —NH—CH₂CH₂—NH— or —NH—CH₂CH₂—O—CH₂CH₂—NH—. Nonlimiting examples of such peptide-linker conducts are:

Alternatively maleimide or pyridine-2-yl-disulfanyl reactive group canbe attached to a polypeptide according to any one of Formula I to IVaccording to scheme 3A, 3B and 3C:

The carboxylic acid group at the C-terminus of the peptide is coupledwith one or more O2Oc amino acid units using standard amide couplingconditions to generate (3A). The terminal carboxylic acid functionalityreacts with the amino group of (3B) or (3C) wherein R and R′ are asdefined above, in order to generate the activated peptide-linkerconstructs (3D) or (3E). Additionally, when a peptide contains a carboxyfunctionality side chain (e.g. Glu or Asp), orthogonal protecting group(e.g. O-Allyl) and additional deprotection steps are required.

Peptide-linker construct 3F can be obtained using a cysteamine2-chlorotrityl Resin and then reacted with 3G or 3H to generatepeptide-linker construct 3I or 3E respectively.

Peptide-linker construct (3J) can be obtained from a diamine resin andbe further reacted with (1B) or (2A) to generate a peptide-linkerconstruct of Formula (3K) or (3L) respectively.Schemes 1 to 3C describe peptide-linker constructs, more particularlyfor use in the preparation of a bioconjugate with Albumin. The maleimidereactive group and the pyridine-2-yl-disulfanyl reactive group reactswith the —SH functionality of Cysteine 34 of the albumin.

Bioconjugates

In one embodiment of the present invention, a peptide or polypeptideaccording to anyone of Formula I to IV is conjugated(chemically/covalently attached) to the thiol functionality of cysteine34 of the albumin. In one embodiment of this embodiment, theAlbumin-Peptide refers to a bioconjugate in which the Albumin isconjugated (chemically linked) to the N-terminus of the peptide. In yetanother embodiment, the Albumin-Peptide refers to a bioconjugate inwhich the Albumin is conjugated (chemically linked) to the C-terminus ofthe peptide.

Peptides covalently linked to Albumin have been found to exhibit asubstantially greater half-life in vivo than the unconjugatedcounterpart.

Preparation of Conjugates:

Schemes 4 and 5 illustrate chemical reactions for conjugation of an APJagonist peptide or a peptide according to anyone of Formula I to IV anda half-life extending moiety such as an Fc domain or albumin.Scheme 4 illustrates the conjugation of a peptide-linker of Formula 4Awith Cysteine 34 of Albumin

wherein L represent a linking moiety between the peptide and themaleimide functionality. In a particular embodiment, L is a linkingmoiety as disclosed in Scheme 1, 3A, 3B or 3C.Scheme 5 illustrates the conjugation of a peptide-linker construct ofFormula 5A with Cysteine 34 of Albumin.

wherein L represents a linking moiety between the peptide and the—S—S-Pyridine functionality. In a particular embodiment, L is a linkingmoiety as disclosed in schemes 2, 3A, 3B or 3C.

Methods for making conjugates and peptide-linker constructs as describedin Schemes 1-5 have also been described and exemplified in co-filedapplication (Attorney docket number: PAT055781-US-PSP) which is herebyincorporated by reference.

Pharmaceutical Compositions

The polypeptides or bioconjugates of the instant invention, or an amide,an ester of a salt thereof, may be administered in any of a variety ofways, including subcutaneously, intramuscularly, intravenously,intraperitoneally, inhalationally, intranasally, orally etc.Particularly preferred embodiments of the invention employ continuousintravenous administration of the polypeptides or bioconjugates of theinstant invention, or an amide, ester, or salt thereof. The polypeptideson the instant invention may be administered as a bolus or as acontinuous infusion over a period of time. An implantable pump may beused. In certain embodiments of the invention, intermittent orcontinuous polypeptides administration is continued for one to severaldays (e.g., 2-3 or more days), or for longer periods of time, e.g.,weeks, months, or years. In some embodiments, intermittent or continuouspolypeptide or bioconjugate administration is provided for at leastabout 3 days. In other embodiments, intermittent or continuouspolypeptide administration is provided for at least about one week. Inother embodiments, intermittent or continuous polypeptide orbioconjugate administration is provided for at least about two weeks. Itmay be desirable to maintain an average plasma polypeptide concentrationabove a particular threshold value either during administration orbetween administration of multiple doses. A desirable concentration maybe determined, for example, based on the subject's physiologicalcondition, disease severity, etc. Such desirable value(s) can beidentified by performing standard clinical trials. Alternatively, thepeptides and conjugates thereof could be delivered orally via FcRnmechanism (Nat Rev Immunol. 7(9), 715-25, 2007; Nat Commun. 3; 3:610,2012, Am J Physiol Gastrointest Liver Physiol 304: G262-G270, 2013).

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a polypeptide or bioconjugate of the presentinvention or and amide, an ester or a salt thereof and one or morepharmaceutically acceptable carriers. The pharmaceutical composition canbe formulated for particular routes of administration such as oraladministration, parenteral administration, and rectal administration,etc. In addition, the pharmaceutical compositions of the presentinvention can be made up in a solid form (including without limitationcapsules, tablets, pills, granules, powders or suppositories), or in aliquid form (including without limitation solutions, suspensions oremulsions). The pharmaceutical compositions can be subjected toconventional pharmaceutical operations such as sterilization and/or cancontain conventional inert diluents, lubricating agents, or bufferingagents, as well as adjuvants, such as preservatives, stabilizers,wetting agents, emulsifiers and buffers, etc.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. Preferred pharmaceutical formulations are stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Ingeneral, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Certain injectable compositions are aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. Said compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1-75%, or contain about 1-50%, of theactive ingredient.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive therapeutic agents arepreferably delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. It is noted that the lungsprovide a large surface area for systemic delivery of therapeuticagents.

The agents may be encapsulated, e.g., in polymeric microparticles suchas those described in U.S. publication 20040096403, or in associationwith any of a wide variety of other drug delivery vehicles that areknown in the art. In other embodiments of the invention the agents aredelivered in association with a charged lipid as described, for example,in U.S. publication 20040062718. It is noted that the latter system hasbeen used for administration of a therapeutic polypeptide, insulin,demonstrating the utility of this system for administration of peptideagents.

Systemic administration can also be by transmucosal or transdermalmeans.

Suitable compositions for transdermal application include an effectiveamount of a polypeptide of the invention with a suitable carrier.Carriers suitable for transdermal delivery include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound of the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Suitable compositions for topical application, e.g., to the skin andeyes, include aqueous solutions, suspensions, ointments, creams, gels orsprayable formulations, e.g., for delivery by aerosol or the like. Suchtopical delivery systems will in particular be appropriate for dermalapplication. They are thus particularly suited for use in topical,including cosmetic, formulations well-known in the art. Such may containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

As used herein a topical application may also pertain to an inhalationor to an intranasal application. They may be conveniently delivered inthe form of a dry powder (either alone, as a mixture, for example a dryblend with lactose, or a mixed component particle, for example withphospholipids) from a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray, atomizer ornebuliser, with or without the use of a suitable propellant.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, etc.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of thepolypeptides of this invention and, which typically are not biologicallyor otherwise undesirable. In many cases, the polypeptides of the presentinvention are capable of forming acid and/or base salts by virtue of thepresence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulfornate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,sulfosalicylic acid, and the like. Pharmaceutically acceptable baseaddition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound, a basic or acidic moiety, byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, use of non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile isdesirable, where practicable. Lists of additional suitable salts can befound, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., MackPublishing Company, Easton, Pa., (1985); and in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

Method of the Invention

Apelin family of peptides is the only known natural family of ligandsfor the G protein coupled APJ receptor. Apelin gene encodes a 77 aminoacid polypeptide, which gets processed into biologically active forms ofapelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13,apelin-12 and pyroglutamate modified form of apelin-13 (Pyr¹-apelin-13).Any one of these apelin peptides, upon binding to APJ receptor,transduces the signal via Gi and Gq proteins. In cardiomyocytes, Gi orGq coupling leads to changes in intracellular pH, PLC activation, andIP3 production that enhance myofilament calcium sensitivity andultimately result in increased cardiac contractility. Gi couplinginhibits activated Gs, adenylyl cyclase and cAMP production andincreases pAkt levels leading to cardioprotection. In vascularendothelial cells, APJ activation via Gi, pAKT leads to increased nitricoxide (NO) production, which increases smooth muscle relaxationresulting in overall vasodilation.

Patients with chronic stable heart failure have occasional acuteepisodes of decompensation, where cardiac contractility declines furtherand symptoms worsen. These exacerbations are referred to as acutedecompensated heart failure (ADHF). Current therapies for ADHF includediuretics, vasodilators, and inotropes, which directly increase cardiaccontractility. Current intravenous inotropes (dobutamine, dopamine,milrinone, levosimendan) are well known for their adverse events such asarrhythmia and increased long-term mortality. The synthetic apelinpolypeptide analogs of the instant invention or bioconjugate thereof,provide a therapy for ADHF that increases cardiac contractility withoutarrhythmogenic or mortality liabilities and address the enormous unmetmedical need in chronic heart failure.

Indeed, acute apelin treatment (5 min) in humans results in coronaryvasodilatation and improved cardiac output. However, native apelinsexhibit a very short t_(1/2) (seconds) and duration of action (fewminutes) in vivo. The potent synthetic apelin peptide agonists of theinstant invention have longer half lives compared to the native apelin.

Activation of APJ receptor in cardiomyocytes a) improve cardiaccontractility via Gi/Gq, PLC and Ca2+, and b) provide cardioprotectionvia Gi, pAkt activation, but without increasing cAMP (as seen with otherinotropes). In addition, APJ agonism in endothelial cells leads toarterial vasodilation, which further benefits heart failure by unloadingthe work of left ventricle. Taken together the synthetic apelinpolypeptide analogs can improve overall cardiac function, reducearrhythmogenesis and provide survival benefit.

More recently, there have been a number of preclinical researchpublications focusing on the potential involvement of Apelin in diabetesand insulin resistance. Apelin has been shown to 1) lower blood glucoselevels by improving glucose uptake in muscle, adipose and heart, 2)protect pancreatic beta cells from ER stress and subsequent apoptosis,3) lower the insulin secretion in beta cells, and 4) regulatecatecholamine induced lypolysis in adipose tissue. Activation of pAKTpathway has been implicated in these processes.

The polypeptides according to anyone of formulae I to IV, or apharmaceutically acceptable salt thereof, or bioconjugates thereof, infree form or in pharmaceutically acceptable salt form, exhibit valuablepharmacological properties, e.g. APJ receptor agonism properties, e.g.as indicated in in vitro and in vivo tests as provided in the nextsections and are therefore indicated for therapy.

Polypeptides of the invention or a pharmaceutically acceptable saltthereof, or bioconjugates thereof, may be useful in the treatment of anindication selected from acute decompensated heart failure (ADHF),chronic heart failure, pulmonary hypertension, atrial fibrillation,Brugada syndrome, ventricular tachycardia, atherosclerosis,hypertension, restenosis, ischemic cardiovascular diseases,cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes(including gestational diabetes), obesity, peripheral arterial disease,cerebrovascular accidents, transient ischemic attacks, traumatic braininjuries, amyotrophic lateral sclerosis, burn injuries (includingsunburn) and preeclampsia.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof or a bioconjugate thereof, for the treatment of a diseasewhich is associated with the APJ receptor activity. In a furtherembodiment, the therapy is selected from a disease which is responsiveto the agonism of the APJ receptor. In another embodiment, the diseaseis selected from the afore-mentioned list, suitably acute decompensatedheart failure. In yet another subset of this embodiment, the presentinvention provides the use of a polypeptide of anyone of formulae I toIV, or an amide, ester or a salt thereof, or a bioconjugate thereof, inthe manufacture of a medicament, for the treatment of a disease which isassociated with the APJ receptor activity.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof, or a bioconjugate thereof, in therapy. In a furtherembodiment, the therapy is selected from a disease which may be treatedby activation (agonism) of the APJ receptor.

In another embodiment, the invention provides a method of treating adisease which is responsive to the agonism of the APJ receptor,comprising administration of a therapeutically acceptable amount of apolypeptide of anyone of formulae I to IV, or an amide, an ester of asalt thereof or a bioconjugate thereof. In a further embodiment, thedisease is selected from the afore-mentioned list, suitably acutedecompensated heart failure.

In yet another subset of this embodiment, the invention provides amethod of treating a disease which is associated with the activity ofthe APJ receptor comprising administration of a therapeuticallyacceptable amount of a polypeptide of anyone of formulae I to IV, or anamide, an ester or a salt thereof or a bioconjugate thereof.

The effective amount of a pharmaceutical composition or combination ofthe invention to be employed therapeutically will depend, for example,upon the therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the fusion protein variant is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the dual function protein in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

In another non-limiting embodiment, the term “a therapeuticallyeffective amount” refers to the amount of the polypeptide of the presentinvention or a bioconjugate thereof, that, when administered to a cell,or a tissue, or a non-cellular biological material, or a medium, iseffective to at least partially activate the APJ receptor. As will beappreciated by those of ordinary skill in the art, the absolute amountof a particular agent that is effective may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the target tissue, etc. Those of ordinary skill in the art understandthat “a therapeutically effective amount” may be administered in asingle dose or may be achieved by administration of multiple doses. Forexample, in the case of an agent to treat heartfailure, an effectiveamount may be an amount sufficient to result in clinical improvement ofthe patient, e.g., increased exercise tolerance/capacity, increasedblood pressure, decrease fluid retention, and/or improved results on aquantitative test of cardiac functioning, e.g., ejection fraction,exercise capacity (time to exhaustion), etc.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, fish, birds and the like. In certain embodiments, the subject is aprimate. In yet other embodiments, the subject is a human.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence, onset, or development of one ormore symptoms of a disorder in a subject resulting from theadministration of a therapy (e.g., a therapeutic agent), or theadministration of a combination of therapies (e.g., a combination oftherapeutic agents).

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The activity of a polypeptide according to the present invention can beassessed by the following in vitro methods described below.

hAPJ Calcium Flux Assay:

Chem-5 APJ stable cells (Millipore # HTS068C) were plated in 384-wellformat with 10,000 cells/well in 25 ul growth media, then grown 24 hoursin a 37° C. tissue culture incubator. One hour before the assay, 25ul/well FLIPR Calcium 4 dye (Molecular Devices R8142) with 2.5 mMprobenecid was added, and cells were incubated one hour in a 37° C.tissue culture incubator. Peptides were solubilized in HBSS, HEPES &0.1% BSA buffer, and serially-diluted 10-fold, from 50 uM to 5 pM, intriplicate. FLIPR Tetra was used to add peptide to the cells with dye(1:5, for final peptide concentrations ranging from 10 uM to 1 pM).FLIPR dye inside the cells emitted fluorescence after binding tocalcium, while fluorescence from outside the cells was masked.Fluorescence was measured using 470-495 excitation and 515-575 emissionwavelengths on the FLIPR Tetra. Readings were done for 3 minutes total,beginning 10 seconds before the peptide addition. Maximum-minimum valueswere calculated and plotted for each peptide concentration, and GraphPad prism software was used to calculate EC₅₀ values at the curveinflection points, for calcium flux stimulation by peptides.

Plasma Stability Assay: Materials:

Working solution: 1 mg/mL test article is prepared in Milli-Q waterExtraction solution: Methanol:Acetonitrile:Water (1:1:1) with 0.1%Formic Acid and 400 ng/mL Glyburide.Plasma: Male Sprague-Dawley rat plasma (with sodium heparin), purchasedfrom Bioreclamation LLC (Liverpool, N.Y.).Whole blood: Male Sprague Dawley whole blood (with sodium heparin),purchased from Bioreclamation LLC (Liverpool, N.Y.)Lung homogenate: Male rat Sprague Dawley lung was purchased fromBioreclamation LLC (Liverpool, N.Y.). The lung was homogenized usingpolytron homogenizer after addition of 5× volume of 1×PBS. Thehomogenate was centrifuged at 9000 rpm for 10 min at 4° C. Thesupernatant was centrifuged again at 3000 rpm for 30 min to make a clearsupernatant. Protein concentration was determined using a commercial kit(Pierce, Thermo Scientific).

Sample Preparation Procedure: (Peptides)

Test article was prepared in one of the following biological matrices:heparinized rat plasma, heparinized rat whole blood or lung homogenate.The plasma and whole blood sample was prepared at 5000 ng/mL by adding 5uL of 1 mg/mL Working solution to 995 uL of rat plasma or whole blood.Lung homogenate samples were prepared by diluting lung homogenate to 1mg/ml protein concentration with phosphate buffered saline (PBS),followed by addition of 5 uL Working solution to 995 uL diluted lunghomogenate. The samples were incubated at 37° C. with gentle shaking(65˜75 rpm) in a water bath incubator. At times 0 min, 5 min, 15 min, 30min, 60 min, 120 and 240 min, 25 uL aliquots of incubation samples weretransferred to 96-well plate and immediately protein precipitated using150 uL of Extraction solution. After completion of incubationexperiment, the sample plate was centrifuged at 4000 rpm at 4° C. for 10minutes. Afterwards, a pipetting device (Tecan Temo) was used totransfer the supernatants to another plate and add 50 uL of water to allsamples. The plate was vortexed prior to LC-MS analysis.

Sample Preparation Procedure (Conjugates)

Test article was prepared at 50,000 ng/mL by adding 5 uL of 1 mg/mLWorking solution to 495 uL of rat plasma. The samples were incubated at37° C. with gentle shaking (65˜75 rpm) in a water bath incubator. Attimes 0 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 and 24 hr, 50 uL aliquots ofincubation samples were transferred to 96-well plate and 100 uL 40 mMTCEP (tris(2-carboxyethyl)phosphine) was added to each sample. Thereaction mixture was incubated at 37° C. for 1 hour. After completion ofreaction, protein precipitation was performed using 300 uL ofacetonitrile. The sample plate was centrifuged at 4000 rpm at 4° C. for10 minutes. Afterwards, a pipetting device (Tecan Temo) was used totransfer 125 uL supernatants to another plate and adds 50 uL of water toall samples. The plate was vortexed prior to LC-MS analysis.

LC-MS Analysis of Stability Samples

HPLC: Agilent 1290 HPLC with autosamplerColumn: MAC-MOD ACE C18, 3 μm, 30 mm×2.1 mm i.d.Mobile phase A: 0.1% Formic acid in acetonitrileMobile phase B: 0.1% Formic acid in water

Gradient Program:

Time (min) Flow (ml.) Mobile Phase A (%) Mobile Phase B (%) 0 0.4 95 50.5 0.4 95 5 1.5 0.4 5 95 4.1 0.4 5 95 4.2 0.4 95 5 5 0.4 95 5Mass spectrometer: Agilent Q-TOF 6530Data acquisition mode: Full scan with mass range of 100-1000 m/zData acquisition and analysis software: MassHunter

Data Analysis:

Stability assay: stability half-life, (t ½), values were determined byconverting peak areas at each time point to percent remaining relativeto initial (t=0) peak area.Percent remaining=100×(sample peak area)÷(t=0 peak area)The natural log of percent remaining values were calculated and plottedagainst sample time (Microsoft Excel). The slope of this line, k, wasdetermined by linear regression (Microsoft Excel).Stability half-life was then calculated by the formula, t ½=0.693÷k

Surrogate Activity-Based Plasma Stability Assay:

The calcium flux protocol described above was followed, with thefollowing changes. The peptides were also incubated with 5% rat plasma(Bioreclamation # RATPLNAHP-M, Na Heparin-treated). Readings were takenat time points t₀ and t₂₄ hrs, after incubation in a 37° C. tissueculture incubator. Peptide plasma half-life in minutes was estimated bycalculating the following:

1) LN((EC₅₀ at t₀)/(EC₅₀ at t_(24 hrs))),

2) Calculate slope of value above and

3) t_(1/2)=0.693/(slopê2).

Using the test assay (as described above) polypeptides of the inventionexhibited efficacy and stability in accordance to Tables 2 and 3,provided infra.

TABLE 2 Activity and stability of polypeptides Surrogate hAPJ Ca²⁺activity-based Flux EC₅₀ Plasma stability Peptide [nM] t½ [min] Example1 2.3 >1000 Example 2 385.7 >1000 Example 3 796.5 >1000 Example 4 5.0730.4 Example 5 79.9 >1000 Example 6 3.1 >1000 Example 7 692.7 >1000Example 8 7.7 >1000 Example 9 9.9 >1000 Example 10 154.6 >1000 Example11 1063.8 >1000 Example 12 0.9 >1000 Example 13 1.1 >1000 Example 14 3.2184.7 Example 15 2.1 >1000 Example 16 8.8 409.0 Example 17 3.1 >1000Example 18 66.5 122.3 Example 19 169.5 >1000 Example 20 922.1 >1000Example 21 0.9 >1000 Example 22 86.8 >1000 Example 23 2.8 124.8 Example24 13.8 57.5 Example 25 69.2 241.5 Example 26 1.7 985.5 Example 27 120.9348.9 Example 28 113.8 >1000 Example 29 1.8 565 Example 30 2.4 128.5Example 31 99.0 346.6 Example 32 30.8 47.7 Example 33 14.8 >1000 Example34 34.5 23.2 Example 35 10.7 >1000 Example 36 16.2 121.4 Example 37 3.9523.5 Example 38 3.3 >1000 Example 39 4.5 888.5 Example 40 2.0 >1000Example 41 4.5 42.2 Example 42 2.4 >1000 Example 43 2.0 178 Example 443.0 555 Example 45 2.4 >1000 Example 46 9.2 532 Example 47 4.4 >1000Example 48 16.1 248 Example 49 623.0 >1000 Example 50 1.4 11 Example 518.5 >1000 Example 52 19.5 >1000 Example 53 8.7 >1000 Example 54 0.9 902Example 55 72.8 101 Example 56 6.8 331 Example 57 1.5 >1000 Example 5855.1 228 Example 59 7.4 849 Example 60 200.0 >1000 Example 61 7.9 >1000Example 62 95.9 474 Example 63 9.4 225 Example 64 30.8 >1000 Comparative1.8 5.0 Example: Pyr1-apelin-13

TABLE 3 Correlation between plasma stability assay and surrogateactivity based plasma stability assay: Plasma Surrogate Activitystability based Plasma Peptide t½ [min] stability t½ [min] Example 4 414730 Example 14 405 185 Example 16 448 409 Example 17 365 >1000 Example29 156 565 Example 30 121 128 Pyr-1-Apelin 13 6.6 5.0

The polypeptide of the present invention or bioconjugate thereof mayhave an APJ receptor potency similar to apelin-13 or pyr-1-apelin-13. Inone embodiment the polypeptide of the present invention or abioconjugate thereof has an EC₅₀ of less than 100 nM. In anotherembodiment the polypeptide of the invention or a bioconjugate thereofhas an EC₅₀ of less than 50 nM, preferably less than 25 nM and morepreferably less than 15 nM. In yet another embodiment, the polypeptideof the present invention has an EC₅₀ of less than 10 nM.

The polypeptide of the present invention or a bioconjugate thereof mayhave plasma stability superior to apelin-13 or pyr-1-apelin-13. In oneembodiment, the plasma stability improvement is at least 2 fold. In oneembodiment, the polypeptide of the invention or a bioconjugate thereofhas a plasma stability of at least 30 minutes. In another embodiment,the polypeptide of the invention or a bioconjugate thereof has a plasmastability of at least 60 minutes, or at least 80 min, preferably atleast 100 minutes and more preferably at least 150 minutes.

The polypeptide of the present invention or a bioconjugate thereof maybe administered either simultaneously with, or before or after, one ormore other therapeutic agent. The polypeptide of the present inventionmay be administered separately, by the same or different route ofadministration, or together in the same pharmaceutical composition asthe other agents.

In one embodiment, the invention provides a product comprising apolypeptide of anyone of formulae I to IV, or an amide, an ester of asalt thereof, or a bioconjugate thereof and at least one othertherapeutic agent as a combined preparation for simultaneous, separateor sequential use in therapy. In one embodiment, the therapy is thetreatment of a disease or condition responsive to the activation of theAPJ receptor.

Products provided as a combined preparation include a compositioncomprising a polypeptide of anyone of formulae I to IV, or an amide, anester of a salt thereof, or a bioconjugate thereof, and the othertherapeutic agent(s) together in the same pharmaceutical composition, ora polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof, or a bioconjugate thereof, and the other therapeuticagent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical compositioncomprising a polypeptide of anyone of formulae I to IV, or an amide, anester or a salt thereof, or a bioconjugate thereof, and anothertherapeutic agent(s). Optionally, the pharmaceutical composition maycomprise a pharmaceutically acceptable excipient, as described above.

In one embodiment, the invention provides a kit comprising two or moreseparate pharmaceutical compositions, at least one of which contains apolypeptide of anyone of formula I to IV, or an amide, an ester or asalt thereof or a bioconjugate thereof. In one embodiment, the kitcomprises means for separately retaining said compositions, such as acontainer, divided bottle, or divided foil packet. An example of such akit is a blister pack, as typically used for the packaging of tablets,capsules and the like.

The kit of the invention may be used for administering different dosageforms, for example, oral and parenteral, for administering the separatecompositions at different dosage intervals, or for titrating theseparate compositions against one another. To assist compliance, the kitof the invention typically comprises directions for administration.

In the combination therapies of the invention, the compound of theinvention and the other therapeutic agent may be manufactured and/orformulated by the same or different manufacturers. Moreover, thecompound of the invention and the other therapeutic may be broughttogether into a combination therapy: (i) prior to release of thecombination product to physicians (e.g. in the case of a kit comprisingthe compound of the invention and the other therapeutic agent); (ii) bythe physician themselves (or under the guidance of the physician)shortly before administration; (iii) in the patient themselves, e.g.during sequential administration of a polypeptide of the invention andthe other therapeutic agent.

Accordingly, the invention provides the use of a polypeptide of anyoneof formulae I to IV, or an amide, an ester or a salt thereof, or abioconjugate thereof, for treating a disease or condition responsive tothe agonism of the APJ receptor, wherein the medicament is prepared foradministration with another therapeutic agent. The invention alsoprovides the use of another therapeutic agent for treating a disease orcondition responsive to the agonism of the apelin receptor, wherein themedicament is administered with a polypeptide of anyone of formulae I toIV, or an amide, an ester or a salt thereof or a bioconjugate thereof.

The invention also provides a polypeptide of anyone of formulae I to IV,or a pharmaceutically acceptable salt thereof or a bioconjugate thereof,for use in a method of treating a disease or condition responsive to theagonism of the APJ receptor, wherein the polypeptide of anyone offormulae I to IV, or an amide, an ester or a salt thereof or abioconjugate thereof, is prepared for administration with anothertherapeutic agent. The invention also provides another therapeutic agentfor use in a method of treating a disease or condition responsive to theagonism of the APJ receptor, wherein the other therapeutic agent isprepared for administration with a polypeptide of anyone of formulae Ito IV, or an amide, an ester or a salt thereof or a bioconjugatethereof. The invention also provides a polypeptide of anyone of formulaeI to IV, or an amide, an ester or a salt thereof, for use in a method oftreating a disease or condition responsive to the agonism of the APJreceptor, wherein the polypeptide of anyone of formulae I to IV, or anamide, an ester or a salt thereof or a bioconjugate thereof, isadministered with another therapeutic agent. The invention also providesanother therapeutic agent for use in a method of treating a disease orcondition responsive to the agonism of the APJ receptor, wherein theother therapeutic agent is administered with a polypeptide of anyone offormulae I to IV or an amide, an ester or a salt thereof or abioconjugate thereof.

The invention also provides the use of a polypeptide of anyone offormulae I to IV, or an amide, an ester or a salt thereof or abioconjugate thereof, for treating a disease or condition responsive tothe agonism of the APJ receptor, wherein the patient has previously(e.g. within 24 hours) been treated with another therapeutic agent. Theinvention also provides the use of another therapeutic agent fortreating a disease or condition responsive to the agonism of the APJreceptor, wherein the patient has previously (e.g. within 24 hours) beentreated with a polypeptide of anyone of formulae I to IV, or an amide,an ester or a salt thereof or a bioconjugate thereof.

In one embodiment, the other therapeutic agent is selected frominotropes, beta adrenergic receptor blockers, HMG-Co-A reductaseinhibitors, angiotensin II receptor antagonists, angiotensin convertingenzyme (ACE) Inhibitors, calcium channel blockers (CCB), endothelinantagonists, renin inhibitors, diuretics, ApoA-I mimics, anti-diabeticagents, obesity-reducing agents, aldosterone receptor blockers,endothelin receptor blockers, aldosterone synthase inhibitors (ASI), aCETP inhibitor, anti-coagulants, relaxin, BNP (nesiritide) and a NEPinhibitor.

The term “in combination with” a second agent or treatment includesco-administration of the polypeptide of the invention (e.g., apolypeptide according to anyone of Formulae I-IV or a bioconjugatethereof or a polypeptide, bioconjugate otherwise described herein) withthe second agent or treatment, administration of the compound of theinvention first, followed by the second agent or treatment andadministration of the second agent or treatment first, followed by thecompound of the invention.

The term “second agent” includes any agent which is known in the art totreat, prevent, or reduce the symptoms of a disease or disorderdescribed herein, e.g. a disorder or disease responsive to theactivation of the APJ receptor, such as for example, acute decompensatedheart failure (ADHF), chronic heart failure, pulmonary hypertension,atrial fibrillation, Brugada syndrome, ventricular tachycardia,atherosclerosis, hypertension, restenosis, ischemic cardiovasculardiseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention,diabetes (including gestational diabetes), obesity, peripheral arterialdisease, cerebrovascular accidents, transient ischemic attacks,traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries(including sunburn) and preeclampsia.

Examples of second agents include inotropes, beta adrenergic receptorblockers, HMG-Co-A reductase inhibitors, angiotensin II receptorantagonists, angiotensin converting enzyme (ACE) Inhibitors, calciumchannel blockers (CCB), endothelin antagonists, renin inhibitors,diuretics, ApoA-I mimics, anti-diabetic agents, obesity-reducing agents,aldosterone receptor blockers, endothelin receptor blockers, aldosteronesynthase inhibitors (ASI), a CETP inhibitor, anti-coagulants, relaxin,BNP (nesiritide) and/or a NEP inhibitor.

Inotropes as used herein include for example dobutamine, isoproterenol,milrinone, amirinone, levosimendan, epinephrine, norepinephrine,isoproterenol and digoxin.

Beta adrenergic receptor blockers as used herein include for exampleacebutolol, atenolol, betaxolol, bisoprolol, carteolol, metoprolol,nadolol, propranolol, sotalol and timolol.

Anti-coagulants as used herein include Dalteparin, Danaparoid,Enoxaparin, Heparin, Tinzaparin, Warfarin.

The term “HMG-Co-A reductase inhibitor” (also calledbeta-hydroxy-beta-methylglutaryl-co-enzyme-A reductase inhibitors)includes active agents that may be used to lower the lipid levelsincluding cholesterol in blood. Examples include atorvastatin,cerivastatin, compactin, dalvastatin, dihydrocompactin, fluindostatin,fluvastatin, lovastatin, pitavastatin, mevastatin, pravastatin,rosuvastatin, rivastatin, simvastatin, and velostatin, or,pharmaceutically acceptable salts thereof.

The term “ACE-inhibitor” (also called angiotensin converting enzymeinhibitors) includes molecules that interrupt the enzymatic degradationof angiotensin I to angiotensin II. Such compounds may be used for theregulation of blood pressure and for the treatment of congestive heartfailure. Examples include alacepril, benazepril, benazeprilat,captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat,fosinopril, imidapril, lisinopril, moexipril, moveltopril, perindopril,quinapril, ramipril, spirapril, temocapril, and trandolapril, or,pharmaceutically acceptables salt thereof.

The term “endothelin antagonist” includes bosentan (cf. EP 526708 A),tezosentan (cf. WO 96/19459), or, pharmaceutically acceptable saltsthereof.

The term “renin inhibitor” includes ditekiren (chemical name:[1S-[1R*,2R*,4R*(1R*,2R*)]]-1-[(1,1-dimethylethoxy)carbonyl]-L-prolyI-L-phenylalanyl-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2-methyl-1-[[(2-pyridinylmrthyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-N-alfa-methyl-L-histidinamide);terlakiren (chemical name:[R—(R*,S*)]-N-(4-morpholinylcarbonyl)-L-phenylalanyl-N-[1-(cyclohexylmethyl)-2-hydroxy-3-(1-methylethoxy)-3-oxopropyl]-S-methyl-L-cysteineamide);Aliskiren (chemical name:(2S,4S,5S,7S)-5-amino-N-(2-carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3-methoxypropoxyl)phenyl]methyl}-8-methyl-2-(propan-2-yl)nonanamide)and zankiren (chemical name:[1S-[1R*[R*(R*)],2S*,3R]]-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5-methylhexy]-alfa-[[2-[[(4-methyl-1-piperazinyl)sulfonyl]methyl]-1-oxo-3-phenylpropyl]-amino]-4-thiazolepropanamide),or, hydrochloride salts thereof, or, SPP630, SPP635 and SPP800 asdeveloped by Speedel, or RO 66-1132 and RO 66-1168 of Formula (A) and(B):

or, pharmaceutically acceptable salts thereof.

The term “aliskiren”, if not defined specifically, is to be understoodboth as the free base and as a salt thereof, especially apharmaceutically acceptable salt thereof, most preferably ahemi-fumarate salt thereof.

The term “calcium channel blocker (CCB)” includes dihydropyridines(DHPs) and non-DHPs (e.g., diltiazem-type and verapamil-type CCBs).Examples include amlodipine, Bepridil, Diltiazem, felodipine, ryosidine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, nitrendipine, Verapamil andnivaldipine, and is preferably a non-DHP representative selected fromthe group consisting of flunarizine, prenylamine, diltiazem, fendiline,gallopamil, mibefradil, anipamil, tiapamil and verapamil, or,pharmaceutically acceptable salts thereof. CCBs may be used asanti-hypertensive, anti-angina pectoris, or anti-arrhythmic drugs.

The term “diuretic” includes thiazide derivatives (e.g., chlorothiazide,hydrochlorothiazide, methylclothiazide, and chlorothalidon).

The term “ApoA-I mimic” includes D4F peptides (e.g., formulaD-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F)

An angiotensin II receptor antagonist or a pharmaceutically acceptablesalt thereof is understood to be an active ingredient which bind to theAT₁-receptor subtype of angiotensin II receptor but do not result inactivation of the receptor. As a consequence of the inhibition of theAT₁ receptor, these antagonists can, for example, be employed asantihypertensives or for treating congestive heart failure.

The class of AT₁ receptor antagonists comprises compounds havingdiffering structural features, essentially preferred are thenon-peptidic ones. For example, mention may be made of the compoundswhich are selected from the group consisting of valsartan, losartan,candesartan, eprosartan, irbesartan, saprisartan, tasosartan,telmisartan, the compound with the designation E-1477 of the followingformula

the compound with the designation SC-52458 of the following formula

and the compound with the designation ZD-8731 of the following formula

or, in each case, a pharmaceutically acceptable salt thereof.

Preferred AT₁-receptor antagonist are candesartan, eprosartan,irbesartan, losartan, telmisartan, valsartan. Also prefered are thoseagents which have been marketed, most preferred is valsartan or apharmaceutically acceptable salt thereof.

The term “anti-diabetic agent” includes insulin secretion enhancers thatpromote the secretion of insulin from pancreatic-cells. Examples includebiguanide derivatives (e.g., metformin), sulfonylureas (SU) (e.g.,tolbutamide, chlorpropamide, tolazamide, acetohexamide,4-chloro-N-[(1-pyrolidinylamino)carbonyl]-benzensulfonamide(glycopyramide), glibenclamide (glyburide), gliclazide,1-butyl-3-metanilylurea, carbutamide, glibonuride, glipizide,gliquidone, glisoxepid, glybuthiazole, glibuzole, glyhexamide,glymidine, glypinamide, phenbutamide, and tolylcyclamide), orpharmaceutically acceptable salts thereof. Further examples includephenylalanine derivatives (e.g., nateglinide[N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine] (cf. EP 196222and EP 526171) of the formula

repaglinide[(S)-2-ethoxy-4-{2-[[3-methyl-1-[2-(1-piperidinyl)phenyl]butyl]amino]-2-oxoethyl}benzoicacid] (cf. EP 589874, EP 147850 A2, in particular Example 11 on page 61,and EP 207331 A1); calcium(2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinlycarbonyl)-propionatedihydrate (e.g., mitiglinide (cf. EP 507534)); and glimepiride (cf. EP31058).

Further examples of second agents with which the peptide and polypeptideof the invention can be used in combination include DPP-IV inhibitors,GLP-1 and GLP-1 agonists.

DPP-IV is responsible for inactivating GLP-1. More particularly, DPP-IVgenerates a GLP-1 receptor antagonist and thereby shortens thephysiological response to GLP-1. GLP-1 is a major stimulator ofpancreatic insulin secretion and has direct beneficial effects onglucose disposal.

The DPP-IV (dipeptidyl peptidase IV) inhibitor can be peptidic or,preferably, non-peptidic. DPP-IV inhibitors are in each case genericallyand specifically disclosed e.g. in WO 98/19998, DE 196 16 486 A1, WO00/34241 and WO 95/15309, in each case in particular in the compoundclaims and the final products of the working examples, thesubject-matter of the final products, the pharmaceutical preparationsand the claims are hereby incorporated into the present application byreference to these publications. Preferred are those compounds that arespecifically disclosed in Example 3 of WO 98/19998 and Example 1 of WO00/34241, respectively.

GLP-1 (glucagon like peptide-1) is an insulinotropic protein which isdescribed, e.g., by W. E. Schmidt et al. in Diabetologia, 28, 1985,704-707 and in U.S. Pat. No. 5,705,483.

The term “GLP-1 agonists” includes variants and analogs ofGLP-1(7-36)NH₂ which are disclosed in particular in U.S. Pat. No.5,120,712, U.S. Pat. No. 5,118,666, U.S. Pat. No. 5,512,549, WO 91/11457and by C. Orskov et al in J. Biol. Chem. 264 (1989) 12826. Furtherexamples include GLP-1(7-37), in which compound the carboxy-terminalamide functionality of Arg³⁶ is displaced with Gly at the 37^(th)position of the GLP-1(7-36)NH₂ molecule and variants and analogs thereofincluding GLN⁹-GLP-1(7-37), D-GLN⁹-GLP-1(7-37), acetyl LYS⁹-GLP-1(7-37),LYS¹⁸-GLP-1(7-37) and, in particular, GLP-1(7-37)OH, VAL⁸-GLP-1(7-37),GLY⁸-GLP-1(7-37), THR⁸-GLP-1(7-37), MET⁸-GLP-1(7-37) and4-imidazopropionyl-GLP-1. Special preference is also given to the GLPagonist analog exendin-4, described by Greig et al. in Diabetologia1999, 42, 45-50.

Also included in the definition “anti-diabetic agent” are insulinsensitivity enhancers which restore impaired insulin receptor functionto reduce insulin resistance and consequently enhance the insulinsensitivity. Examples include hypoglycemic thiazolidinedione derivatives(e.g., glitazone,(S)-((3,4-dihydro-2-(phenyl-methyl)-2H-1-benzopyran-6-yl)methyl-thiazolidine-2,4-dione(englitazone),5-{[4-(3-(5-methyl-2-phenyl-4-oxazolyl)-1-oxopropyl)-phenyl]-methyl}-thiazolidine-2,4-dione(darglitazone),5-{[4-(1-methyl-cyclohexyl)methoxyphenyl]methyl}-thiazolidine-2,4-dione(ciglitazone),5-{[4-(2-(1-indolyl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(DRF2189),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy)]benzyl}-thiazolidine-2,4-dione(BM-13.1246), 5-(2-naphthylsulfonyl)-thiazolidine-2,4-dione (AY-31637),bis{4-[(2,4-dioxo-5-thiazolidinyl)methyl]phenyl}methane (YM268),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl}-thiazolidine-2,4-dione(AD-5075),5-[4-(1-phenyl-1-cyclopropanecarbonylamino)-benzyl]-thiazolidine-2,4-dione(DN-108)5-{[4-(2-(2,3-dihydroindol-1-yl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione,5-[3-(4-chloro-phenyl])-2-propynyl]-5-phenylsulfonyl)thiazolidine-2,4-dione,5-[3-(4-chlorophenyl])-2-propynyl]-5-(4-fluorophenyl-sulfonyl)thiazolidine-2,4-dione,5-{[4-(2-(methyl-2-pyridinyl-amino)-ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(rosiglitazone),5-{[4-(2-(5-ethyl-2-pyridyl)ethoxy)phenyl]-methyl}thiazolidine-2,4-dione(pioglitazone),5-{[4-((3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy)-phenyl]-methyl}-thiazolidine-2,4-dione(troglitazone),5-[6-(2-fluoro-benzyloxy)naphthalen-2-ylmethyl]-thiazolidine-2,4-dione(MCC555),5-{[2-(2-naphthyl)-benzoxazol-5-yl]-methyl}thiazolidine-2,4-dione(T-174) and5-(2,4-dioxothiazolidin-5-ylmethyl)-2-methoxy-N-(4-trifluoromethyl-benzyl)benzamide(KRP297)).

Further anti-diabetic agents include, insulin signalling pathwaymodulators, like inhibitors of protein tyrosine phosphatases (PTPases),antidiabetic non-small molecule mimetic compounds and inhibitors ofglutamine-fructose-6-phosphate amidotransferase (GFAT); compoundsinfluencing a dysregulated hepatic glucose production, like inhibitorsof glucose-6-phosphatase (G6Pase), inhibitors offructose-1,6-bisphosphatase (F-1,6-Bpase), inhibitors of glycogenphosphorylase (GP), glucagon receptor antagonists and inhibitors ofphosphoenolpyruvate carboxykinase (PEPCK); pyruvate dehydrogenase kinase(PDHK) inhibitors; inhibitors of gastric emptying; insulin; inhibitorsof GSK-3; retinoid X receptor (RXR) agonists; agonists of Beta-3 AR;agonists of uncoupling proteins (UCPs); non-glitazone type PPARγagonists; dual PPARα/PPARγ agonists; antidiabetic vanadium containingcompounds; incretin hormones, like glucagon-like peptide-1 (GLP-1) andGLP-1 agonists; beta-cell imidazoline receptor antagonists; miglitol;α₂-adrenergic antagonists; and pharmaceutically acceptable saltsthereof.

In one embodiment, the invention provides a combination, in particular apharmaceutical combination, comprising a therapeutically effectiveamount of the polypeptide according to the definition of anyone offormulae I to IV, or an amide, an ester or a salt thereof, or abioconjugate thereof, and one or more therapeutically active agentsselected from β-adrenergic receptor blockers such as acebutolol,atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol,sotalol and timolol; angiotensin II receptor antagonists such as AT1blockers; antidiabetic agents such as DPPIV inhibitors (e.g.vildagliptin) and GLP1 peptide agonist.

The term “obesity-reducing agent” includes lipase inhibitors (e.g.,orlistat) and appetite suppressants (e.g., sibutramine and phentermine).

An aldosterone synthase inhibitor or a pharmaceutically acceptable saltthereof is understood to be an active ingredient that has the propertyto inhibit the production of aldosterone. Aldosterone synthase (CYP11B2)is a mitochondrial cytochrome P450 enzyme catalyzing the last step ofaldosterone production in the adrenal cortex, i.e., the conversion of11-deoxycorticosterone to aldosterone. The inhibition of the aldosteroneproduction with so-called aldosterone synthase inhibitors is known to bea successful variant to treatment of hypokalemia, hypertension,congestive heart failure, atrial fibrillation or renal failure. Suchaldosterone synthase inhibition activity is readily determined by thoseskilled in the art according to standard assays (e.g., US 2007/0049616).

The class of aldosterone synthase inhibitors comprises both steroidaland non-steroidal aldosterone synthase inhibitors, the later being mostpreferred.

Preference is given to commercially available aldosterone synthaseinhibitors or those aldosterone synthase inhibitors that have beenapproved by the health authorities.

The class of aldosterone synthase inhibitors comprises compounds havingdiffering structural features. An example of non-steroidal aldosteronesynthase inhibitor is the (+)-enantiomer of the hydrochloride offadrozole (U.S. Pat. Nos. 4,617,307 and 4,889,861) of formula

or, if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. inUS2007/0049616, in particular in the compound claims and the finalproducts of the working examples, the subject-matter of the finalproducts, the pharmaceutical preparations and the claims are herebyincorporated into the present application by reference to thispublication. Preferred aldosterone synthase inhibitors suitable for usein the present invention include, without limitation4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-methylbenzonitrile;5-(2-chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid (4-methoxybenzyl)methylamide;4′-fluoro-6-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid butyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;5-(2-Chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 4-fluorobenzyl ester;5-(4-Cyano-2-trifluoromethoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid methyl ester;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 2-isopropoxyethyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methylbenzonitrile;4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;3-Fluoro-4-(7-methylene-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)benzonitrile;cis-3-Fluoro-4-[7-(4-fluoro-benzyl)-5,6,7,8-tetrahydro-imidazo[1,5-a]pyridin-5-yl]benzonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrileor in each case, the (R) or (S) enantiomer thereof; or if appropriable,a pharmaceutically acceptable salt thereof.

The term aldosterone synthase inhibitors also include compounds andanalogs disclosed in WO2008/076860, WO2008/076336, WO2008/076862,WO2008/027284, WO2004/046145, WO2004/014914, WO2001/076574.

Furthermore Aldosterone synthase inhibitors also include compounds andanalogs disclosed in U.S. patent applications US200710225232,US2007/0208035, US2008/0318978, US2008/0076794, US2009/0012068,US20090048241 and in PCT applications WO2006/005726, WO2006/128853,WO2006128851, WO2006/128852, WO2007065942, WO2007/116099, WO2007/116908,WO2008/119744 and in European patent application EP 1886695. Preferredaldosterone synthase inhibitors suitable for use in the presentinvention include, without limitation8-(4-Fluorophenyl)-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazine;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-fluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2,6-difluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-methoxybenzonitrile;3-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)phthalonitrile;4-(8-(4-Cyanophenyl)-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)naphthalene-1-carbonitrile;8-[4-(1H-Tetrazol-5-yl)phenyl1-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazineas developed by Speedel or in each case, the (R) or (S) enantiomerthereof; or if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. in WO2009/156462 and WO 2010/130796, in particular in the compound claims andthe final products of the working examples, the subject-matter of thefinal products, the pharmaceutical preparations and the claims.Preferred Aldosterone Synthase inhibitors suitable for combination inthe present invention include,3-(6-Fluoro-3-methyl-2-pyridin-3-yl-1H-indol-1-ylmethyl)-benzonitrilehydrochloride,1-(4-Methanesulfonyl-benzyl)-3-methyl-2-pyridin-3-yl-1H-indole,2-(5-Benzyloxy-pyridin-3-yl)-6-chloro-1-methyl-1H-indole,5-(3-Cyano-1-methyl-1H-indol-2-yl)-nicotinic acid ethyl ester,N-[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,Pyrrolidine-1-sulfonic acid5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl ester,N-Methyl-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,6-Chloro-1-methyl-2-{5-[(2-pyrrolidin-1-yl-ethylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,6-Chloro-2-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-pyridin-3-yl]-1-methyl-1H-indole-3-carbonitrile,6-Chloro-1-methyl-2-{5-[(1-methyl-piperidin-4-ylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,Morpholine-4-carboxylic acid[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-amide,N-[5-(6-Chloro-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,C,C,C-Trifluoro-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-4-trifluoromethyl-benzenesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-1-phenyl-methanesulfonamide,N-(5-(3-chloro-4-cyanophenyl)pyridin-3-yl)butane-1-sulfonamide,N-(1-(5-(4-cyano-3-methoxyphenyl)pyridin-3-yl)ethyl)ethanesulfonamide,N-((5-(3-chloro-4-cyanophenyl)pyridin-3-yl)(cyclopropyl)methyl)ethanesulfonamide,N-(cyclopropyl(5-(1H-indol-5-yl)pyridin-3-yl)methyl)ethanesulfonamide,N-(cyclopropyl(5-naphtalen-1-yl-pyridin-3-yl)methyl)ethanesulfonamide,Ethanesulfonic acid[5-(6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-pyridin-3-ylmethyl]-amideand Ethanesulfonic acid{[5-(3-chloro-4-cyano-phenyl)-pyridin-3-yl]-cyclopropyl-methyl}-ethyl-amide.

The term “endothelin receptor blocker” includes bosentan andambrisentan.

The term “CETP inhibitor” refers to a compound that inhibits thecholesteryl ester transfer protein (CETP) mediated transport of variouscholesteryl esters and triglycerides from HDL to LDL and VLDL. Such CETPinhibition activity is readily determined by those skilled in the artaccording to standard assays (e.g., U.S. Pat. No. 6,140,343). Examplesinclude compounds disclosed in U.S. Pat. No. 6,140,343 and U.S. Pat. No.6,197,786 (e.g.,[2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylicacid ethyl ester (torcetrapib); compounds disclosed in U.S. Pat. No.6,723,752 (e.g.,(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol);compounds disclosed in U.S. patent application Ser. No. 10/807,838;polypeptide derivatives disclosed in U.S. Pat. No. 5,512,548;rosenonolactone derivatives and phosphate-containing analogs ofcholesteryl ester disclosed in J. Antibiot., 49(8): 815-816 (1996), andBioorg. Med. Chem. Lett.; 6:1951-1954 (1996), respectively. Furthermore,the CETP inhibitors also include those disclosed in WO2000/017165,WO2005/095409, WO2005/097806, WO 2007/128568, WO2008/009435, WO2009/059943 and WO2009/071509.

The term “NEP inhibitor” refers to a compound that inhibits neutralendopeptidase (NEP) EC 3.4.24.11. Examples include Candoxatril,Candoxatrilat, Dexecadotril, Ecadotril, Racecadotril, Sampatrilat,Fasidotril, Omapatrilat, Gemopatrilat, Daglutril, SCH-42495, SCH-32615,UK-447841, AVE-0848, PL-37 and(2R,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoicacid ethyl ester or a pharmaceutically acceptable salt thereof. NEPinhibitors also include Phosphono/biaryl substituted dipeptidederivatives, as disclosed in U.S. Pat. No. 5,155,100. NEP inhibitorsalso include N-mercaptoacyl phenylalanine derivative as disclosed in PCTapplication Number WO 2003/104200. NEP inhibitors also includedual-acting antihypertensive agents as disclosed in PCT applicationNumbers WO 2008/133896, WO 2009/035543 or WO 2009/134741. Other examplesinclude compounds disclosed in U.S. application Ser. Nos. 12/788,794;12/788,766 and 12/947,029. NEP inhibitors also include compoundsdisclosed in WO 2010/136474, WO 2010/136493, WO 2011/061271 and USprovisional applications Nos. 61/414,171 and 61/414,163.

In one embodiment, the invention provides a method of activating the APJreceptor in a subject, wherein the method comprises administering to thesubject a therapeutically effective amount of the polypeptide accordingto the definition of anyone of formulae I to IV, or an amide, an esteror a salt thereof, or a bioconjugate thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation of the APJ receptor,in a subject, wherein the method comprises administering to the subjecta therapeutically effective amount of the polypeptide according to thedefinition of anyone of formulae I to IV, or an amide, an ester or asalt thereof or a bioconjugate thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation (agonism) of the APJreceptor, in a subject, wherein the disorder or the disease is selectedfrom acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

In one embodiment, the invention provides a polypeptide according to thedefinition of anyone of formulae I to IV or a bioconjugate thereof, foruse as a medicament.

In one embodiment, the invention provides the use of a polypeptideaccording to the definition of anyone of formulae I to IV, or an amide,an ester or a salt thereof or a bioconjugate thereof, in the manufactureof a medicament, for the treatment of a disorder or disease responsiveto the activation of the APJ receptor. In another embodiment, theinvention provides the use of a polypeptide according to the definitionof anyone of formulae I to IV, or an amide, an ester or a salt thereofor a bioconjugate thereof, in the manufacture of a medicament, for thetreatment of a disorder or disease responsive to the activation of theAPJ receptor, wherein said disorder or disease is in particular selectedfrom acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

Exemplification of the Invention Peptide and Polypeptide Synthesis

Abbreviation Definition AA Amino acid Ac Acetyl Acm Acetamidomethyl ACNAcetonitrile AcOH Acetic acid Ac₂O Acetic anhydride ε-Ahx ε-Aminohexanoic acid AM Aminomethyl BAL Backbone amide linker BSA Bovine SerumAlbumin Boc tert-Butyloxycarbonyl Bzl Benzyl DCM Dichlormethane DICN,N′-Diisopropylcarbodiimide DIPEA N,N′-Diisopropylethylamine DMAN,N′-Dimethylacetamide DMF N,N′-Dimethylformamide DMSO DimethylsulfoxideDTT Dithiothreitol DVB Divinylbenzene EDT Ethanedithiol FA Formic acidFmoc 9-Fluorenylmethyloxycarbonyl HATU2-(1H-9-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate HBSS Hank's buffered salt solution HCTU2-(6-Chloro-1H-Benzotriazole-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid HFIP Hexafluoroisopropanol HOAt 1-Hydroxy-7-azabenzotriazole HPLCHigh performance liquid chromatography HSA Human albumin serum ivDde(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LN Logarithmusnaturali (natural logarithm) MeOH Methanol MS Mass spectrometry Nal2-Naphthylalanine Nle Norleucine NMP N-Methylpyrrolidine Oxyma PureEthyl 2-cyano-2-(hydroxyimino)acetate Pbf2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl PBS Phosphate bufferedsaline pE Pyroglutamate PhP Phenylproline Pip Pipecolic acid PGProtecting group Ph Phenyl Pol Polymer support PS Polystyrene rt Roomtemperature SEC Size-exclusion chromatography SPPS Solid phase peptidesynthesis tBuOH tert-Butanol TCEP Tris(2-carboxyethyl)phosphine TFATrifluoroacetic acid THF Tetrahydrofuran TIS or TIPS TriisopropylsilaneTPA 3-Mercaptopropanoic Acid t_(R) Retention time Trt Trityl UPLC Ultraperformance liquid chromatography UV Ultraviolet

The peptides were synthesized by standard solid phase Fmoc chemistry.The peptides were assembled on the Prelude™ peptide synthesizer (ProteinTechnologies, Inc., Tucson, USA) and Liberty microwave peptidesynthesizer (CEM Corporation, North Carolina, USA). Peptides with a freecarboxylic acid on the C-terminus were synthesized from 2-chlorotritylchloride-PS-resin (ABCR, Karlsruhe, Germany or AnaSpec, Inc.,California, USA). Peptides with an unsubstituted carboxamide on theC-terminus were synthesized from Fmoc protected Rink-Amide-AM-PS-resin(Merck, Darmstadt, Germany). Peptides with an N-monosubstitutedcarboxamide on the C-terminus were synthesized from BAL-AM-PS-resinloaded with amines (EMC Microcollections, Tübingen, Germany).

The peptides were purified by preparative reversed-phase HPLC. Thefollowing columns were used:

-   -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×100 mm, Part No.        186002572 (one column or two columns in series)    -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×150 mm, Part No.        186002797    -   Waters Atlantis Prep OBD T3 Column, 5 μm, 30×150 mm, Part No.        186003703    -   Waters XBridge Prep C8 OBD Column, 5 μm, 30×150 mm, Part No.        186003083    -   Machery-Nagel Nucleosil® 100-5 C18, 5 μm, 250×40 mm, Part No.        715340.400

Mobile phases consisted of eluent A (0.1% TFA in H₂O) and eluent B(ACN). Gradients were designed based on the specific requirements of theseparation problem. Pure products were lyophilized from ACN/H₂O.

The products were analyzed by analytical HPLC using UV detection atλ=214 nm and UPLC-MS using electrospray ionization.

The peptides that are exemplified in Table 4 were synthesized using thegeneral procedures described below. Unsubstituted N- or C-termini areindicated by italic H— or —OH, respectively.

TABLE 4 Example Sequence Example 1 pE-R-P-R-L-a-H-K-G-Pip-nle-a-f-OHExample 2 pE-R-P-R-(D-Leu)-S-a-K-G-P-nle-a-f-OH Example 3pE-r-P-R-L-a-H-K-G-P-nle-a-f-OH Example 4pE-R-P-R-(D-Leu)-S-Aib-K-G-P-nle-a-f-OH Example 5pE-R-P-R-L-S-Aib-k-G-P-nle-a-f-OH Example 6pE-R-P-R-L-a-H-K-G-P-nle-f-OH Example 7pE-R-P-R-L-c(tBu)-H-K-G-P-nle-a-f-OH Example 8pE-R-P-R-L-d-H-K-G-P-nle-a-f-OH Example 9pE-R-(trans-4-PhP)-R-L-a-H-K-G-P-nle-a-f-OH Example 10pE-R-P-R-L-(D-Leu)-H-K-G-P-nle-a-f-OH Example 11pE-R-P-R-L-k-H-K-G-P-nle-a-f-OH Example 12pE-R-P-R-L-s-H-K-G-P-nle-a-f-OH Example 13pE-R-P-R-L-a-H-K-G-P-nle-p-f-OH Example 14pE-R-P-R-L-a-H-K-G-P-nle-(D-Leu)-f-OH Example 15pE-R-P-R-L-a-Aib-K-G-P-nle-a-f-OH Example 16pE-R-P-R-L-a-h-K-G-P-nle-a-f-OH Example 17pE-R-P-R-L-a-F-K-G-P-nle-a-f-OH Example 18 pE-R-P-R-L-a-H-K-G-P-nle-F-OHExample 19 pE-R-P-R-L-a-H-K-G-P-nle-e-f-OH Example 20pE-R-P-R-L-a-H-K-G-P-nle-r-f-OH Example 21pE-R-P-R-L-a-H-K-G-P-nle-a-y-OH Example 22pE-R-P-R-L-a-H-K-G-P-nle-a-d-OH Example 23pE-R-P-R-Cha-a-H-K-G-P-nle-a-f-OH Example 24pE-R-P-R-(3,4-Cl2-F)-a-H-K-G-P-nle-a-f-OH Example 25pE-R-P-R-(2-Nal)-a-H-K-G-P-nle-a-f-OH Example 26pE-R-P-R-L-a-H-K-G-P-nle-a-tic-OH Example 27pE-R-P-R-Y(Bzl)-a-H-K-G-P-nle-a-f-OH Example 28pE-R-P-R-Y-a-H-K-G-P-nle-a-f-OH Example 29pE-R-P-R-L-a-H-K-G-P-nle-a-f-OH Example 30pE-R-P-R-L-a-H-K-G-P-Nle-a-f-OH Example 31pE-R-P-R-L-a-H-k-G-P-Nle-a-f-OH Example 32pE-R-P-R-L-a-H-K-G-P-nle-NH(Phenethyl) Example 33pE-R-P-R-L-a-H-K-G-P-nle-a-f-NH2 Example 34pE-R-P-R-L-a-H-K-G-P-Nle-P-f-OH Example 35 pE-R-P-R-L-a-H-K-G-P-Nle-f-OHExample 36 pE-R-P-R-L-a-H-K-G-P-nle-a-NH(Phenethyl) Example 37pE-R-P-R-L-S-Aib-K-G-P-nle-a-F-OH Example 38pE-R-P-R-L-S-Aib-K-G-P-Nle-a-f-OH Example 39pE-R-P-R-L-a-H-K-G-P-Nle-a-F-OH Example 40pE-R-P-R-L-S-Aib-K-G-P-nle-a-f-OH Example 41pE-R-P-R-L-S-Aib-K-G-P-Nle-P-f-OH Example 42pE-R-P-R-L-S-Aib-K-G-P-Nle-a-F-OH Example 43pE-R-P-R-L-a-H-K-G-P-nle-(4-Phenoxypiperidin-1-yl) Example 44pE-R-P-R-L-abu-H-K-G-P-nle-a-f-OH Example 45pE-R-P-R-L-a-H-K-G-P-nle-abu-f-OH Example 46pE-R-P-R-L-a-f-K-G-P-nle-a-f-OH Example 47pE-R-P-R-L-a-L-K-G-P-nle-a-f-OH Example 48pE-R-P-R-L-a-a-K-G-P-nle-a-f-OH Example 49pE-R-a-R-L-a-H-K-G-P-nle-a-f-OH Example 50 H-R-P-R-L-a-H-K-G-P-nle-f-OHExample 51 pE-R-P-R-L-S-a-K-G-P-nle-a-f-OH Example 52pE-R-P-R-L-nva-H-K-G-P-nle-a-f-OH Example 53pE-R-P-R-L-a-H-K-G-P-nva-a-f-OH Example 54pE-R-P-R-L-a-H-K-G-P-nle-nva-f-OH Example 55pE-R-P-R-L-S-f-K-G-P-nle-a-f-OH Example 56pE-R-P-R-L-S-h-K-G-P-nle-a-f-OH Example 57 pE-R-P-R-L-a-H-K-G-P-f-a-f-OHExample 58 pE-R-P-R-L-A-h-K-G-P-nle-a-f-OH Example 59pE-R-P-R-L-a-H-Q-G-P-nle-a-f-OH Example 60pE-R-P-R-L-a-H-E-G-P-nle-a-f-OH Example 61pE-R-P-R-L-v-H-K-G-P-nle-a-f-OH Example 62pE-R-P-R-L-a-H-K-D-P-nle-a-f-OH Example 63pE-R-P-R-Cha-nva-H-K-G-P-nle-a-f-OH

Analytical Methods 1a) HPLC—Analytical Method A

-   -   Column: Bischoff UHC-640 (53×4.0 mm) with ProntoSil 120-3-C18-H,        3 μm; Part n°: 0604F185PS030    -   Eluent A: 0.07% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.5 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 95 5 10 0 100 12.0 0 100 12.2 95 5

1b) HPLC—Analytical Method B

-   -   Column: Bischoff UHC-640 (53×4.0 mm) with ProntoSil 120-3-C18-H,        3 μm; Part n°: 0604F185PS030    -   Eluent A: 0.1% TFA in water/Eluent B: 0.4% TFA in ACN/Eluent C:        MeOH    -   Flow: 1.5 ml/min    -   Temperature: 25° C.    -   Gradient:

Time [min] A [%] B [%] C [%] 0.0 90 2.5 7.5 9.5 0 25 75 12.0 0 25 7512.2 90 2.5 7.5

1c) HPLC—Analytical Method C

-   -   Column: XBridge BEH300 C18 (100×4.6 mm), 3 μm; Part n°:        186003612    -   Eluent A: 0.1% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 98 2 18 2 98 20 2 98 22 98 2

2) UPLC-MS—Analytic Method D

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.1% FA in water; Eluent B: 0.1% FA in ACN    -   Flow: 0.7 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 99 1 1.0 97 3 3.5 50 50 4.0 10 90 4.3 0 1004.6 80 20

3) UPLC-HRMS—Analytic Method E

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.1% FA; Eluent B: 0.1% FA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 50° C.    -   Gradient: 2 to 98% in 4.4 min

4) HPLC—Analytical Method F

-   -   Column: YMC-Gel ODS-A-C18    -   Eluent A: 0.1% v/v TFA in Water/Eluent B: ACN/Eluent A, 9/1 v/v    -   Elution with gradient:

Time Flux A B [min] [ml/min] [% v/v] [% v/v] 0 50 95 5 10 50 95 5 40 5080 20 45 50 80 20 55 50 75 25 65 50 75 25 75 50 70 30 105 50 70 30

5) UPLC-MS—Analytical Method G

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.05% FA+3.75 mM ammonium acetate in water; Eluent B:        0.04% FA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 50° C.    -   Gradient: 2 to 44% in 1.7 min

6) UPLC-HRMS—Analytic Method H

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.05% FA+3.75 mM ammonium acetate in water; Eluent B:        0.04% FA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 50° C.    -   Gradient: 2 to 98% in 4.4 min

4) UPLC-MS—Analytic Method I

-   -   Waters Acquity UPLC® BEH300 SEC guard column, 4.6×30 mm; Part        n°: 186005793    -   Eluent A: 0.1% FA in water; Eluent B: 0.04% FA in ACN    -   Flow: 1.0 ml/min    -   Gradient: 50% B for 6 min

5) UPLC-MS—Analytic Method J

-   -   Waters Acquity UPLC® ProSwift RP-3, 1.7 μm, 4.6×50 mm; Part n°:        064298    -   Eluent A: 0.1% FA in water; Eluent B: 0.08% FA in ACN    -   Flow: 2.0 ml/min (3 to 80% B in 2 min)-flow 1.8 mL/min    -   Temperature: 40° C.    -   Gradient: 2 to 98% in 3 min        The analytical data for peptides of Examples 1 to 63 are        summarized in Table 5 and was generated using the analytical        methods described supra.

TABLE 5 Mass spectrometry HPLC [M + 2H]²⁺ [M + 3H]³⁺ [M + 2H]²⁺ [M +3H]³⁺ Peptide t_(R) [min] Meth. (measured) (measured) Meth. (calc.)(calc.) Example 1 3.43 A 496.7 D 744.4 496.6 Example 2 3.40 A 712.3475.1 D 712.4 475.3 Example 3 3.28 A 491.9 D 737.4 491.9 Example 4 3.40A 719.2 479.9 D 719.4 479.9 Example 5 3.44 A 719.2 479.7 D 719.4 479.9Example 6 3.40 A 468.2 D 701.9 468.3 Example 7 3.69 A 521.2 D 781.4521.3 Example 8 3.24 A 506.5 D 759.4 506.6 Example 9 3.65 A 517.2 D775.4 517.3 Example 10 3.58 A 505.7 D 758.4 506.0 Example 11 3.13 A510.8 D 765.9 511.0 Example 12 3.26 A 497.1 D 745.4 497.3 Example 133.43 A 750.2 500.3 D 750.4 500.6 Example 14 3.59 A 505.7 D 758.4 506.0Example 15 3.57 A 711.1 474.5 D 711.4 474.6 Example 16 3.40 A 491.7 D737.4 491.9 Example 17 3.88 A 742.2 495.1 D 742.4 495.3 Example 18 3.43A 468.1 D 701.9 468.3 Example 19 3.27 A 766.4 D 766.4 511.3 Example 203.02 A 520.2 D 779.9 520.3 Example 21 2.82 A 745.3 D 745.4 497.3 Example22 2.49 A 481.2 D 721.4 481.3 Example 23 3.52 A 505.2 D 757.4 505.3Example 24 3.65 A 526.0 D 788.4 525.9 Example 25 3.57 A 779.4 519.8 D779.4 519.9 Example 26 3.24 A 495.9 D 743.4 496.0 Example 27 3.89 A807.3 D 807.4 538.6 Example 28 3.12 A 508.5 D 762.4 508.6 Example 293.35 A 491.8 D 737.4 491.9 Example 30 5.03 B 491.7 D 737.4 491.9 Example31 5.22 B 737.4 491.9 D 737.4 491.9 Example 32 5.47 B 679.9 453.6 D679.8 453.6 Example 33 3.17 A 736.7 491.6 D 736.9 491.6 Example 34 3.37A 500.6 D 750.4 500.6 Example 35 3.28 A 468.2 D 701.9 468.3 Example 363.41 A 477.2 D 715.4 477.3 Example 37 3.59 A 719.3 479.9 D 719.4 479.9Example 38 3.41 A 719.4 479.9 D 719.4 479.9 Example 39 3.27 A 491.9 D737.4 491.9 Example 40 3.48 A 719.2 479.9 D 719.4 479.9 Example 41 3.53A 732.4 488.6 D 732.4 488.6 Example 42 3.49 A 719.3 479.9 D 719.4 479.9Example 43 3.93 A 708.6 472.6 D 707.9 472.3 Example 44 7.04 C 744.4496.6 E 744.9 496.9 Example 45 7.17 C 744.4 496.6 E 744.9 496.9 Example46 7.95 C 742.4 495.3 E 742.9 495.6 Example 47 7.98 C 725.4 484.0 E725.9 484.2 Example 48 7.52 C 704.4 470.0 E 704.8 470.2 Example 49 7.04C 724.4 483.3 E 724.8 483.6 Example 50 6.77 C 646.4 431.3 E 646.8 431.5Example 51 6.79 C 712.4 475.3 E 712.8 475.6 Example 52 7.49 C 751.4501.3 E 751.9 501.6 Example 53 6.46 C 730.4 487.3 E 730.8 487.6 Example54 7.50 C 751.4 501.3 E 751.9 501.6 Example 55 7.67 C 750.4 500.6 E750.9 500.9 Example 56 6.93 C 745.4 497.3 E 745.9 497.6 Example 57 7.16C 754.4 503.3 E 754.9 503.6 Example 58 7.01 C 737.4 492.0 E 737.9 492.2Example 59 7.37 C 737.4 492.0 E 737.9 492.2 Example 60 7.42 C 737.9492.3 E 738.3 492.6 Example 61 7.33 C 751.4 501.3 E 751.9 501.6 Example62 7.33 C 766.4 511.3 E 766.9 511.6 Example 63 8.16 C 771.5 514.6 E771.9 514.9

General Synthesis Procedures

1) Loading of First Amino Acid onto 2-Chlorotrityl Chloride Resin andFmoc-Removal

2-Chlorotrityl chloride resin (1 eq., 1.0-1.6 mmol/g) was washedthoroughly with DCM. The desired amino acid (typically 0.5-2 eq.relative to the resin, considering 1.6 mmol/g loading) was dissolved inDCM (approx. 10 mL per gram of resin) and DIPEA (4 eq. relative to theresin, considering 1.6 mmol/g loading). The solution was added to theresin and the suspension was shaken at rt for 19 h. The resin wasdrained and then thoroughly washed sequentially with DCM/MeOH/DIPEA(17:2:1), DCM, DMA, DCM.

For Fmoc removal and determination of the loading the resin was shakenrepeatedly with piperidine/DMA (1:4) or 4-methylpiperidine/DMA (1:4)(12×10 mL per gram of initial resin) and washed with DMA (2×10 mL pergram of initial resin). The combined solutions were diluted with MeOH toa volume V of 250 mL per gram of initial resin. A 2 mL aliquot (V_(a))of this solution was diluted further to 250 mL (V_(t)) with MeOH. The UVabsorption was measured at 299.8 nm against a reference of MeOH, givingabsorption A. The resin was thoroughly washed sequentially with DMA,DCM, DMA, DCM and dried in high vacuum at 40° C., affording m g ofresin.

The loading of the resin is calculated according to the formula:

Loading[mol/g]=(A×V _(t) ×V)/(d×ε×V _(a) ×m)

(with d: width of cuvette; ε=7800 L mol⁻¹ cm⁻¹)

2) Solid Phase Peptide Synthesis 2a) Synthesis Cycle a on Prelude™Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith 4-methylpiperidine/DMA (1:4). The resin was washed with DMA.Coupling was done by addition of the Fmoc-amino acid (3 eq.; 0.2 Msolution in NMP), HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (3.3eq.; 0.66 M solution in NMP) followed by mixing of the suspension withnitrogen at rt for typically 15 min to 4 h depending on the specificrequirements. After washing with DMA the coupling step was typicallyrepeated 1 to 3 times depending on the specific requirements. Afterwashing with DMA capping was performed by addition of a mixture ofAc₂O/pyridine/DMA (1:1:8) and subsequent mixing of the suspension at rt.The resin was washed with DMA.

2b) Synthesis Cycle B on Prelude™ Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4). The resin was washed with DMA. Coupling wasdone by addition of the Fmoc-amino acid (3 eq.; 0.3 M solution in NMP),HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (4.5 eq.; 0.9 M solutionin NMP) followed by mixing of the suspension with nitrogen at rt fortypically 15 min to 4 h depending on the specific requirements. Afterwashing with DMA the coupling step was typically repeated 1 to 3 timesdepending on the specific requirements. After washing with DMA cappingwas performed by addition of a mixture of Ac₂O/pyridine/DMA (1:1:8) andsubsequent mixing of the suspension at rt. The resin was washed withDMA.

2c) Synthesis Cycle C on Liberty™ Synthesizer

The resin was washed with DMF and DCM. Fmoc was removed by treatmentwith 20% piperidine/DMF (typically 7 ml per 0.1 mmol twice). The resinwas washed with DMF and DCM. Coupling was done by addition of theFmoc-amino acid (5 eq.; 0.2 M solution in DMF), HCTU (5 eq.; 0.5 Msolution in DMF), and DIPEA (10 eq.; 2 M solution in NMP) followed bymixing of the suspension with nitrogen at 75 or 50° C. for typically 5to 50 min with microwave power 0 to 20 watts depending on the specificrequirements. After washing with DMF the coupling step might be repeatedonce depending on the specific requirements. The resin was washed withDMF.

3) Cleavage from Resin with or without Concomitant Removal of ProtectingGroups

3a) Cleavage Method A

The resin (0.1 mmol) was shaken at rt for typically 1.5-2 h with 95% aq.TFA/EDT/TIS (95:2.5:2.5) (2 mL). The cleavage solution was filtered off,and fresh solution was added (2 mL). The suspension was shaken at rt fortypically 0.75-1 h then the cleavage solution was filtered off. Freshsolution was added (2 mL) and the suspension was shaken at rt fortypically 0.75-1 h. The cleavage solution was filtered off. The resinwas rinsed once with 95% aq. TFA (1 mL). The combined cleavage andwashing solutions were poured slowly onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum.

3b) Cleavage Method B

The resin (0.1 mmol) was shaken at rt for 1.5 h with 95% aq. TFA/TIS(97.5:2.5) (2 mL). The cleavage solution was filtered off, and freshsolution was added (2 mL). The suspension was shaken at rt for 45 minthen the cleavage solution was filtered off. Fresh solution was added (2mL) and the suspension was shaken at rt for 45 min. The cleavagesolution was filtered off. The resin was rinsed once with 95% aq. TFA (1mL). The combined cleavage and washing solutions were poured slowly ontoa mixture of cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (10mL), the suspension was centrifuged and the supernatant was poured off.The solid was dried in high vacuum.

3c) Cleavage Method C

HFIP/DCM (10:90) (2 mL) was added to the resin (0.1 mmol) and thesuspension was shaken at rt for 10 min. The cleavage solution wasfiltered off into iPrOH (0.8 mL). This step was repeated 3 times,combining the cleavage solutions with the first iPrOH-containingcleavage solution directly. The combined cleavage solutions wereconcentrated to dryness in high vacuum. The residue was lyophilized fromtBuOH/H₂O (4:1).

3d) Cleavage Method D

The resin (0.1 mmol) was shaken at rt for 3 h with 95% aq. TFA//TIS/DTT(95:2.5:2.5) (3 mL). The cleavage solution was filtered off. The resinwas rinsed once with 95% aq. TFA (1 mL). The combined cleavage andwashing solutions were poured slowly onto a mixture of coldheptane/diethyl ether (1:1) (10-15 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. Diethyl ether(10 mL) was added to the residue, the suspension was vortexed for 3 minand centrifuged, and the supernatant was poured off, The wash processwas repeated twice. The solid was dried in high vacuum.

In the following the syntheses of representative examples are described.

Example 1 Synthesis of pE-R-P-R-L-a-H-K-G-Pip-nle-a-f-OH

Example 1 Preparation of Intermediate 1a Loading of 2-ChlorotritylChloride Resin with Fmoc-f-OH, Fmoc Removal and Determination of theLoading of the Resin

2-Chlorotrityl chloride resin (3.0 g, 4.80 mmol) was reacted with asolution of Fmoc-f-OH (1.86 g, 4.80 mmol) in DCM (30 mL) and DIPEA (3.35mL, 19.2 mmol) in analogy to the general procedure described above togive Intermediate 1a (3.53 g, loading=0.96 mmol/g).

Preparation of Intermediate 1b Assembly of Linear Peptide

Intermediate 1a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 a 2 ×15 min A 2 nle 2 × 15 min A 3 Pip 2 × 15 min A 4 G 2 × 30 min A 5 K(Boc)2 × 15 min A 6 H(Trt) 2 × 15 min A 7 a 2 × 15 min A 8 L 2 × 15 min A 9R(Pbf) 4 × 1 h A 10 P 2 × 15 min A 11 R(Pbf) 4 × 1 h A 12 pE 2 × 15 minA

Preparation of Example 1 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL) was added toIntermediate 1b (0.1 mmol) and the suspension was shaken at rt for 1.5h. The cleavage solution was filtered off, and fresh cleavage solution(2 mL) was added. The suspension was shaken at rt for 45 min then thecleavage solution was filtered off. Fresh solution (2 mL) was added andthe suspension was shaken at rt for 45 min. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage and washing solutions were poured onto a mixture ofcold heptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 1 (86.5 mg, 0.044 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.43 min) and UPLC-MS (Analytical method D; measured:[M+3H]³⁺=496.7; calculated: [M+3H]³⁺=496.6).

Example 29 Synthesis pE-R-P-R-L-a-H-K-G-P-nle-a-f-OH

Example 29 Preparation of Intermediate 29a Loading of 2-ChlorotritylChloride Resin with Fmoc-f-OH, Fmoc Removal and Determination of theLoading of the Resin

2-Chlorotrityl chloride resin (2 g, 3.20 mmol) was reacted with asolution of Fmoc-f-OH (992 mg, 2.56 mmol) in DCM (20 mL) and DIPEA(2.236 mL, 12.80 mmol) in analogy to the general procedure describedabove to give Intermediate 29a (2.36 g, loading=0.82 mmol/g).

Preparation of Intermediate 29b Assembly of Linear Peptide

Intermediate 29a (0.600 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 a 2 ×15 min B 2 nle 2 × 15 min B 3 P 2 × 15 min B 4 G 2 × 30 min B 5 K(Boc) 2× 15 min B 6 H(Trt) 2 × 15 min B 7 a 2 × 15 min B 8 L 2 × 15 min B 9R(Pbf) 4 × 1 h B 10 P 2 × 15 min B 11 R(Pbf) 4 × 1 h B 12 pE 2 × 15 minB

Preparation of Example 29 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (10 mL) was added toIntermediate 29b (0.6 mmol) and the suspension was shaken at rt for 2 h.The cleavage solution was filtered off, and fresh cleavage solution (10mL) was added. The suspension was shaken at rt for 1 h then the cleavagesolution was filtered off. Fresh solution (10 mL) was added and thesuspension was shaken at rt for 1 h. The cleavage solution was filteredoff. The combined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (200 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (100 mL), thesuspension was centrifuged and the supernatant was poured off. The solidwas dried in high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 29 (538.7 mg, 0.279 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.35 min) and UPLC-MS (Analytical method D; measured:[M+3H]³⁺=491.8; calculated: [M+3H]³⁺=491.9).

Example 32 Synthesis of pE-R-P-R-L-a-H-K-G-P-nle-NH(Phenethyl)

Example 32 Preparation of Intermediate 32a Assembly of Linear Peptide

Phenethylamine-BAL-PS resin (167 mg, 0.100 mmol) was subjected to solidphase peptide synthesis on the Prelude™ peptide synthesizer. Couplingwas performed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 nle 2× 30 min B 2 P 2 × 30 min B 3 G 3 × 1 h B 4 K(Boc) 2 × 30 min B 5 H(Trt)2 × 30 min B 6 a 2 × 30 min B 7 L 2 × 30 min B 8 R(Pbf) 4 × 1 h B 9 P 2× 90 min B 10 R(Pbf) 4 × 1 h B 11 pE 2 × 90 min B

Preparation of Example 32 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL) was added toIntermediate 32a (0.1 mmol) and the suspension was shaken at rt for 1.5h. The cleavage solution was filtered off, and fresh cleavage solution(2 mL) was added. The suspension was shaken at rt for 1.5 h then thecleavage solution was filtered off. Fresh solution (2 mL) was added andthe suspension was shaken at rt for 2 h. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (5 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 32 (37.0 mg, 0.020 mmol).

The pure product was analyzed by analytical HPLC (Analytical method B:t_(R)=5.47 min) and UPLC-MS (Analytical method D; measured:[M+3H]³⁺=453.6; calculated: [M+3H]³⁺=453.6).

Example 33 Synthesis of pE-R-P-R-L-a-H-K-G-P-nle-a-f-NH₂

Example 33 Preparation of Intermediate 33a Assembly of Linear Peptide

Fmoc protected Rink-Amide-AM-PS-resin (217 mg, 0.100 mmol) was subjectedto solid phase peptide synthesis on the Prelude™ peptide synthesizer.Coupling was performed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 f 2 ×15 min A 2 a 2 × 1 h A 3 nle 2 × 15 min A 4 P 2 × 15 min A 5 G 2 × 30min A 6 K(Boc) 2 × 15 min A 7 H(Trt) 2 × 15 min A 8 a 2 × 15 min A 9 L 2× 15 min A 10 R(Pbf) 4 × 1 h A 11 P 2 × 30 min A 12 R(Pbf) 4 × 1 h A 13pE 2 × 30 min A

Preparation of Example 33 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/TIS (97.5:2.5) (2 mL) was added to Intermediate33a (0.1 mmol) and the suspension was shaken at rt for 1.5 h. Thecleavage solution was filtered off, and fresh cleavage solution (2 mL)was added. The suspension was shaken at rt for 45 min then the cleavagesolution was filtered off. Fresh solution (2 mL) was added and thesuspension was shaken at rt for 45 min. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 33 (108.9 mg, 0.055 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.17 min) and UPLC-MS (Analytical method D; measured:[M+3H]³⁺=491.6; calculated: [M+3H]³⁺=491.6).

Example 43 Synthesis pE-R-P-R-L-a-H-K-G-P-nle-(4-Phenoxypiperidin-1-yl)

Example 43 Preparation of Intermediate 43a Loading of 2-ChlorotritylChloride Resin with Fmoc-Nle-OH, Fmoc Removal and Determination of theLoading of the Resin

2-Chlorotrityl chloride resin (300 mg, 0.48 mmol) was reacted with asolution of Fmoc-Nle-OH (136 mg, 0.384 mmol) in DCM (3 mL) and DIPEA(0.335 mL, 1.92 mmol) in analogy to the general procedure describedabove to give Intermediate 43a (329 mg, loading=0.99 mmol/g).

Preparation of Intermediate 43b Assembly of Linear Peptide

Intermediate 43a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 P 2 ×15 min A 2 G 2 × 30 min A 3 K(Boc) 2 × 15 min A 4 H(Trt) 2 × 15 min A 5a 2 × 15 min A 6 L 2 × 15 min A 7 R(Pbf) 4 × 1 h A 8 P 2 × 30 min A 9R(Pbf) 4 × 1 h A 10 pE 2 × 30 min A

Preparation of Intermediate 43c HFIP Cleavage from the Resin

HFIP/DCM (10:90) (2 mL) was added to Intermediate 43b (0.100 mmol) andthe suspension was stirred at rt for 10 min. The cleavage solution wasfiltered off into iPrOH (0.8 mL). This step was repeated 3 times,combining the cleavage solutions with the first iPrOH-containingcleavage solution directly. The combined cleavage solutions wereconcentrated to dryness in high vacuum. The residue was lyophilized fromtBuOH/H₂O (4:1) to give Intermediate 43c.

Preparation of Intermediate 43d Coupling of 4-phenoxypiperidine

A mixture of Intermediate 43c with HATU (49.4 mg, 0.130 mmol), HOAt(17.7 mg, 0.130 mmol) and 2,6-lutidine (0.233 mL, 2.000 mmol) in NMP (5mL) was stirred at rt for 5 min. 4-Phenoxypiperidine (35.0 mg, 0.197mmol) was added and stirring was continued at rt for 45 min. Thereaction mixture was concentrated to dryness in vacuo to giveIntermediate 43d.

Preparation of Example 43 Removal of Protecting Groups and Purification

Intermediate 43d was dissolved in 95% aq. TFA/EDT/TIS (95:2.5:2.5) (5mL) and the solution was stirred at rt for 2 h. The cleavage solutionwas poured onto cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (5mL), the suspension was centrifuged and the supernatant poured off. Theresidue was dried in high vacuum. The product was isolated bypreparative HPLC and lyophilized from ACN/H₂O to afford Example 43 (19.3mg, 0.010 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.93 min) and UPLC-MS (Analytical method D; measured:[M+3H]³⁺=472.6; calculated: [M+3H]³⁺=472.3).

Example 57 Synthesis of pE-R-P-R-L-a-H-K-G-P-f-a-f-OH

Example 57 Preparation of Intermediate 57a Loading of 2-ChlorotritylChloride Resin with Fmoc-f-OH, Fmoc Removal and Determination of theLoading of the Resin

2-Chlorotrityl chloride resin (5.0 g, 8.01 mmol) was reacted with asolution of Fmoc-f-OH (3.10 g, 8.01 mmol) in DCM (50 mL) and DIPEA (5.59mL, 32.0 mmol) in analogy to the general procedure described above togive Intermediate 57a (5.87 g, loading=0.897 mmol/g).

Preparation of Intermediate 57b Assembly of Linear Peptide

Intermediate 57a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Liberty™ microwave peptide synthesizer. Coupling wasperformed as follows:

Number of Temper- Micro- couplings × ature wave Synthesis Coupling AAReaction time ° C. power cycle 1 a 1 × 7.5 min 50 20 C 2 f 1 × 7.5 min50 20 C 3 P 1 × 7.5 min 50 20 C 4 G 1 × 7.5 min 50 20 C 5 K(Boc) 1 × 7.5min 50 20 C 6 H(Trt) 1 × 2 min 50 0 C 1 × 4 min 50 25 7 a 1 × 7.5 min 5025 C 8 L 1 × 7.5 min 50 25 C 9 R(Pbf) 2 × 42 min 50 0 C 2 × 7.5 min 5025 10 P 1 × 7.5 min 50 25 C 11 R(Pbf) 2 × 42 min 50 0 C 2 × 7.5 min 5025 12 pE 1 × 7.5 min 50 25 C

Preparation of Example 57 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/EDT/DTT (95:2.5:2.5) (3 mL) was added toIntermediate 57b (0.1 mmol) and the suspension was shaken at rt for 3 h.The cleavage solution was filtered off and the resin was washed with 95%aq. TFA (1 mL). The combined cleavage and washing solutions were pouredonto a mixture of cold heptane/diethyl ether (1:1) (11 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. Diethyl ether (10 mL) was added to the residue, the suspension wasvortexed for 3 min and centrifuged, and the supernatant was poured off,The wash process was repeated twice. The solid was dried in high vacuumThe crude was purified by preparative HPLC and lyophilized from ACN/H₂Oto afford Example 57 (59 mg, 0.030 mmol).

The pure product was analyzed by analytical HPLC (Analytical method C:t_(R)=7.16 min) and UPLC-MS (Analytical method E; measured:[M+3H]³⁺=503.3; calculated: [M+3H]3+=503.6).

This crude isolated TFA salt was dissolved in water (15 mL water/mmol ofpolypeptide) and was converted into the acetate salt using an ionexchanger resin of the type strong base in its hydroxide form [Ionexchanger III, strong base, (OH form. 0.9 mmol/ml), Merck-Darmstadt,Germany, Catalog number; 1.04767.0500, (25 eq)], and the pure acetatesalt product was isolated via lyophilization.

The salt stoichiometry was evaluated based on the analysis of the aceticcontent (ion chromatography) and water content and was determined torange between 1:3 and 1:4 (polypeptide:acetate).

The other examples were synthesized in analogy:

-   -   Examples 2 to 28 were synthesized in analogy to Example 1.    -   Example 30 and 31 were synthesized in analogy to Example 29.    -   Example 34 to 42 were synthesized in analogy to Example 33.    -   Example 44 to 56 and 58 to 63 were synthesized in analogy to        Example 57.

Example 64 Albumin-TPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-R-L-a-H-K-G-P-f-a-f-OHwherein TPA: 3-mercaptopropanoic acid Step 1: Synthesis of Intermediate64c (PPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-R-L-a-H-K-G-P-f-a-f-OH)

Preparation of Intermediate 64a Assembly of Linear Peptide

H-f-O-PS (1.0 mmol) was subjected to solid phase peptide synthesis onthe Liberty™ microwave peptide synthesizer. Coupling was performed asfollows:

Number of Temper- Micro- couplings × ature wave Synthesis Coupling AAReaction time ° C. power cycle 1 a 1 × 7.5 min 50 20 C 2 f 1 × 7.5 min50 20 C 3 P 1 × 7.5 min 50 20 C 4 G 1 × 7.5 min 50 20 C 5 K(Boc) 1 × 7.5min 50 20 C 6 H(Trt) 1 × 2 min 50 0 C 1 × 4 min 50 25 7 a 1 × 7.5 min 5020 C 8 L 1 × 7.5 min 50 25 C 9 R(Pbf) 2 × 42 min 50 0 C 2 × 7.5 min 5025 10 P 1 × 7.5 min 50 25 C 11 R(Pbf) 2 × 42 min 50 0 C 2 × 7.5 min 5025 12 Q(Trt) 1 × 7.5 min 50 25 C 13 O2Oc 1 × 7.5 min 50 25 C 14 O2Oc 1 ×7.5 min 50 25 C 15 O2Oc 1 × 7.5 min 50 25 C 16 O2Oc 1 × 7.5 min 50 25 C17 (Trt)TPA 1 × 7.5 min 50 25 C

Preparation of Intermediate 64b Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A solution made of 3.09 g of DTT and 3 mL of thioanisole in 25 mL ofTFA/TIPS/Water (95:2.5:2.5) was added to Intermediate 64a (1.0 mmol) andthe suspension was shaken at rt for 3 hr. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (5 mL). Thecombined cleavage and washing solutions were poured onto cold diethylether (80 mL), giving a precipitate. The suspension was centrifuged andthe supernatant poured off. Diethyl ether (80 mL) was added to theresidue. The suspension was vortexed for 3 min, centrifuged, and thesupernatant was poured off. The washing process was repeated 3 threetimes. The solid was dried in high vacuum The crude was purified bypreparative HPLC and lyophilized from ACN/H₂O to afford Intermediate 64bas a white powder (235 mg, 84 μmol).

The pure product was analyzed by UPLC-MS (Analytical method G; measured:[M+2H]²⁺=1097.5; calculated: [M+2H]²⁺=1097.8).

Preparation of Intermediate 64cPPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-R-L-a-H-K-G-P-f-a-f-OH Wherein PPA:3-(2-pyridyldithio)propionic acid

A mixture of Intermediate 64b (76 mg, 29 μmol), 2,2′-dithiodipyridine(19 mg, 86 μmol) in ACN (1 mL) was shaked at 25° C. for 1 hr, Thereaction mixture was diluted with MeOH and filtered. The solution waspurified by preparative HPLC and lyophilized from ACN/H2O to affordIntermediate 64c as a white powder (28 mg, 10 μmol).

The pure product was analyzed by UPLC-MS (Analytical method G; measured:[M+2]²⁺=1152.5; calculated: [M+2]²⁺=1152.3).

Step 2: Albumin Decapping Decapping with TCEP

To a solution of albumin (500 mg, Aldrich, lyophilized powder, fromhuman serum) in 10 mL of PBS 1× buffer in a 15 mL tube was added asolution of TCEP hydrochloride (1.074 mg in bio-grade purified water)once. The resultant solution was shaked at Rt for 1 hr, then desaltedand washed with two Amicon Ultra-4 centrifugal filters (30K MWCO). Thefilters were spinned at 4K g for 40 mins and the filtrate werediscarded. 3 mL of bio-grade purified water was added to each filter foreach wash (spinned at 14K g for 10 mins) and the wash process wasrepeated 3 times. The decapped HSA was dissolved in water (˜20 mL intotal). The solution was transferred to a 50 mL Falcon tube, andlyophilized to give a crystalline powder (500 mg).

The pure product was analyzed by UPLC-MS (Analytical method I; measured:66439.0; expected: 66437).

Determination of the Number of Free Thiol Group in Decapped HSA

To a solution of this decapped HSA (2 mg) in 400 μL of PBS pH 7.4 in a 2mL tube was added a solution of 6-maleimidohexanoic acid (13 μg) inwater. The resultant solution was shaked at Rt for 2 hr. UPLC-MS(Analytical method J) showed mono-adduct formation only, measured:66649.0; expected: 66648.

Decapping with DTT

To a solution of albumin (400 mg, Aldrich, lyophilized powder, fromhuman serum) in 5 mL of PBS 1× buffer in a 15 mL tube was added asolution of DTT (0.232 μl, 2 mg/mL in bio-grade purified water) once.The resultant solution was shaked at Rt for 2 hrs, then desalted andwashed with twenty Amicon Ultra-0.5 centrifugal filters (10K MWCO). Thefilters were spinned at 14K g for 10 mins and the filtrates werediscarded. Bio-grade purified water was added to the top of each filterfor each wash (spinned at 14K g for 10 mins) and the wash process wasrepeated 6 times. The decapped HSA was dissolved in water (˜20 mL intotal). The solution was transferred to a 50 mL Falcon tube, andlyophilized to give a crystalline powder (376 mg).

The pure product was analyzed by UPLC-MS (Analytical method J; measured:66438.5; expected: 66437).

Determination of the Number of Free Thiol Group in Decapped HSA

To a solution of this decapped HSA (3 mg) in 400 μL of PBS pH 7.4 in a 2mL tube was added a solution of 3-maleimidopropionic acid (25 μg) inwater. The resultant solution was shaked at Rt overnight. UPLC-MS(Analytical method J) showed mono-adduct formation only, measured:66608.0; expected: 66606.

Decapping with Cysteine

To a solution of albumin (120 mg, Aldrich, lyophilized powder, fromhuman serum) in 1 mL of 50 mM PBS buffer pH 8.0 in a 2 mL tube was addedcysteine (10.94 mg) once. The resultant solution was shaked at Rt for 1hr, then desalted and washed with two Amicon Ultra-0.5 centrifugalfilters (10K MWCO). The filters were spinned at 14K g for 10 mins andthe filtrates were discarded. Bio-grade purified water was added to thetop of each filter for each wash (spinned at 14K g for 10 mins) and thewash process was repeated 5 times. The decapped HSA was dissolved inwater (4 mL in total). The solution was transferred to a 15 mL Falcontube, and lyophilized to give a crystalline powder (108 mg).

The pure product was analyzed by UPLC-MS (Analytical method J; measured:66439; expected: 66437).

Determination of the Number of Free Thiol Group in Decapped HSA

To a solution of this decapped HSA (3 mg) in 500 μL of PBS pH 7.4 in a 2mL tube was added a solution of 3-maleimidopropionic acid (15 μg) inwater. The resultant solution was shaked at Rt for 1 hr. UPLC-MS(Analytical method J) showed mono-adduct formation only, measured:66608.0; expected: 66606.

Step 3: Albumin-TPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-R-L-a-H-K-G-P-f-a-f-OH

A solution of decapped HSA (100 mg) in PBS buffer (6 mL) was treatedwith a solution of Intermediate 64c (7.8 mg in water). The resultantsolution was shaked at Rt for 1 hr, then desalted and washed with 4Amicon Ultra-0.5 centrifugal filters (10K MWCO). The filters werespinned at 13K g for 10 mins and the filtrates were discarded. Bio-gradepurified water was added to the top of each filter for each wash(spinned at 13K g for 10 mins) and the wash process was repeated 6times. The conjugate was dissolved in water (4 mL in total). Thesolution is transferred to a 15 mL Falcon tube, and lyophilized to givea crystalline powder (90.5 mg).

The pure product was analyzed by UPLC-MS (Analytical method I; measured:68630; expected: 68629).

The polypeptide in the examples supra have been found to have EC₅₀,values in the range of about 0.01 nM to about 1100 nM for APJ receptorpotency. The polypeptides in the examples below have been found to havea plasma stability higher than 2 minutes, higher than 5 minutes, higherthan 10 minutes, higher than 20 minutes, higher than 50 minutes andhigher than 60 minutes.

It can be seen that the polypeptides of the invention or bioconjugatesthereof are useful as agonist of the APJ receptor and therefore usefulin the treatment of diseases and conditions responsive to the activationof the APJ receptor, such as the diseases disclosed herein.

Furthermore, half-life of these peptides have been shown to be furtherextended by forming a bioconjugate comprising a peptide or polypeptideaccording to anyone of Formula I to IV with a half-life extendingmoiety, such as Human Serum Albumin.

Having thus described exemplary embodiments of the present invention, itshould be noted by those of ordinary skill in the art that the withindisclosures are exemplary only and that various other alternatives,adaptations, and modifications may be made within the scope of thepresent invention. Accordingly, the present invention is not limited tothe specific embodiments as illustrated therein.

1. A bioconjugate or multimer thereof comprising: a. a peptide or apolypeptide having the following formula (SEQ ID NO: 1):X1-X2-X3-R-X5-X6-X7-X8-X9-X10-X11-X12-X13  I wherein: X1 is theN-terminus of the polypeptide and is either absent, Q, A or pE; X2 is Ror r; X3 is P or 4-PhP; X5 is L, Cha, D-L, F, Y, Y(Bzl), 3,4-Cl2-F orNal; X6 is a D-amino acid, S or A; X7 is a D-amino acid, L, H or Aib;and at least one of X6 and X7 is D-amino acid or Aib; X8 is K, k, Q orE; X9 is G or D; X10 is P or pipecolic acid; X11 is D-Nle, Nle, f orD-Nva; X12 is absent, P or a D-amino acid; X13 is the C-terminus and isabsent, F or a D-amino acid; and at least one of X11, X12 and X13 is aD-amino acid; wherein: Nle is L-norleucine; D-Nle is D-norleucine; Nalis L-naphthyl)alanine; D-Nva is D-norvaline; Aib is α-aminoisobutyricacid; Cha is (S)-β-cyclohexylalanine; D-Tic isD-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; pE is L-pyroglutamicacid; 3,4-Cl2-F is (S)-3,4-dichlorophenylalanine; Y is L-tyrosine; andY(Bzl) is L-benzyl-tyrosine; or an amide, an ester or a salt of thepolypeptide; and b. a half-life extending moiety; wherein said peptideor polypeptide and half-life extending moiety are covalently linked,optionally via a linker.
 2. The bioconjugate of claim 1 wherein thepeptide or polypeptide is of Formula I and X6 and X12 are D-amino acids(SEQ ID NO: 4); or an amide, an ester or a salt thereof. 3-4. (canceled)5. The bioconjugate of claim 1 wherein the peptide or polypeptide is ofFormula I and X6 and X13 are D-amino acids (SEQ ID NO: 7); or an amide,an ester or a salt thereof.
 6. The bioconjugate of claim 1 wherein thepeptide or polypeptide is of Formula I and X7 and X12 are D-amino acids(SEQ ID NO: 8); or an amide, an ester or a salt thereof.
 7. (canceled)8. The bioconjugate according to claim 1 wherein the peptide orpolypeptide has the following formula (SEQ ID NO: 10):X1-R-P-R-X5-a-X7-X8-G-P-X11-X12-X13  II or an amide, an ester or a saltof the polypeptide.
 9. The bioconjugate according to claim 1 wherein thepeptide or polypeptide has Formula III (SEQ ID NO: 11):X1-R-P-R-X5-X6-X7-K-G-P-X11-a-f  III; or an amide, an ester or a salt ofthe polypeptide.
 10. The bioconjugate according to claim 1 wherein thepeptide or polypeptide has Formula IV (SEQ ID NO: 12):X1-R-P-R-X5-S-X7-K-G-P-X11-X12-X13  IV; or an amide, an ester or a saltof the polypeptide.
 11. The bioconjugate peptide according to claim 1wherein the peptide or polypeptide is of Formula I and X6 is a D-aminoacid selected from a, D-Leu, k, s, d, nva, abu, f, h, v and D-Cys(tBu)(SEQ ID NO: 13); or an amide, an ester or a salt thereof.
 12. Thebioconjugate according to claim 1 wherein the peptide or polypeptide isof Formula I and X7 is Aib or a D-amino acid selected from a, f and h(SEQ ID NO: 14); or an amide, an ester or a salt thereof.
 13. Thebioconjugate of claim 1 wherein the peptide or polypeptide is of FormulaI and X12 is absent or a D-amino acid selected from a, f, p, e, r, abu,nva, and D-Leu (SEQ ID NO: 15); or an amide, an ester or a salt thereof.14. The bioconjugate of claim 1 wherein the peptide or polypeptide is ofFormula I and X13 is absent or is a D-amino acid selected from f, y, d,and D-Tic (SEQ ID NO: 16), or an amide, an ester or a salt thereof.15-21. (canceled)
 22. The bioconjugate according to claim 1 wherein thepeptide or polypeptide is selected from: (SEQ ID NO: 24)pE-R-P-R-L-a-H-K-G-Pip-nle-a-f-OH  (SEQ ID NO: 25)pE-R-P-R-(D-Leu)-S-a-K-G-P-nle-a-f-OH  (SEQ ID NO: 26)pE-r-P-R-L-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 27)pE-R-P-R-(D-Leu)-S-Aib-K-G-P-nle-a-f-OH  (SEQ ID NO: 28)pE-R-P-R-L-S-Aib-k-G-P-nle-a-f-OH  (SEQ ID NO: 29)pE-R-P-R-L-a-H-K-G-P-nle-f-OH  (SEQ ID NO: 30)pE-R-P-R-L-c(tBu)-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 31)pE-R-P-R-L-d-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 32)pE-R-(trans-4-PhP)-R-L-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 33)pE-R-P-R-L-(D-Leu)-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 34)pE-R-P-R-L-k-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 35)pE-R-P-R-L-s-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 36)pE-R-P-R-L-a-H-K-G-P-nle-p-f-OH  (SEQ ID NO: 37)pE-R-P-R-L-a-H-K-G-P-nle-(D-Leu)-f-OH  (SEQ ID NO: 38)pE-R-P-R-L-a-Aib-K-G-P-nle-a-f-OH  (SEQ ID NO: 39)pE-R-P-R-L-a-h-K-G-P-nle-a-f-OH  (SEQ ID NO: 40)pE-R-P-R-L-a-F-K-G-P-nle-a-f-OH  (SEQ ID NO: 41)pE-R-P-R-L-a-H-K-G-P-nle-F-OH  (SEQ ID NO: 42)pE-R-P-R-L-a-H-K-G-P-nle-e-f-OH  (SEQ ID NO: 43)pE-R-P-R-L-a-H-K-G-P-nle-r-f-OH  (SEQ ID NO: 44)pE-R-P-R-L-a-H-K-G-P-nle-a-y-OH  (SEQ ID NO: 45)pE-R-P-R-L-a-H-K-G-P-nle-a-d-OH  (SEQ ID NO: 46)pE-R-P-R-Cha-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 47)pE-R-P-R-(3,4-Cl₂-F)-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 48)pE-R-P-R-(2-NaI)-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 49)pE-R-P-R-L-a-H-K-G-P-nle-a-tic-OH  (SEQ ID NO: 50)pE-R-P-R-Y(BzI)-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 51)pE-R-P-R-Y-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 52)pE-R-P-R-L-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 53)pE-R-P-R-L-a-H-K-G-P-Nle-a-f-OH  (SEQ ID NO: 54)pE-R-P-R-L-a-H-k-G-P-Nle-a-f-OH  (SEQ ID NO: 55)pE-R-P-R-L-a-H-K-G-P-nle-NH(Phenethyl)  (SEQ ID NO: 56)pE-R-P-R-L-a-H-K-G-P-nle-a-f-NH2  (SEQ ID NO: 57)pE-R-P-R-L-a-H-K-G-P-Nle-P-f-OH  (SEQ ID NO: 58)pE-R-P-R-L-a-H-K-G-P-Nle-f-OH  (SEQ ID NO: 59)pE-R-P-R-L-a-H-K-G-P-nle-a-NH(Phenethyl)  (SEQ ID NO: 60)pE-R-P-R-L-S-Aib-K-G-P-nle-a-F-OH  (SEQ ID NO: 61)pE-R-P-R-L-S-Aib-K-G-P-Nle-a-f-OH  (SEQ ID NO: 62)pE-R-P-R-L-a-H-K-G-P-Nle-a-F-OH  (SEQ ID NO: 63)pE-R-P-R-L-S-Aib-K-G-P-nle-a-f-OH  (SEQ ID NO: 64)pE-R-P-R-L-S-Aib-K-G-P-Nle-P-f-OH  (SEQ ID NO: 65)pE-R-P-R-L-S-Aib-K-G-P-Nle-a-F-OH  (SEQ ID NO: 66)pE-R-P-R-L-a-H-K-G-P-nle-(4-Phenoxypipendin-1-yl)  (SEQ ID NO: 67)pE-R-P-R-L-abu-H-K-G-P-nle-a-f-OH (SEQ ID NO: 68)pE-R-P-R-L-a-H-K-G-P-nle-abu-f-OH  (SEQ ID NO: 69)pE-R-P-R-L-a-f-K-G-P-nle-a-f-OH  (SEQ ID NO: 70)pE-R-P-R-L-a-L-K-G-P-nle-a-f-OH  (SEQ ID NO: 71)pE-R-P-R-L-a-a-K-G-P-nle-a-f-OH  (SEQ ID NO: 72)pE-R-a-R-L-a-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 73) H-R-P-R-L-a-H-K-G-P-nle-f-OH  (SEQ ID NO: 74)pE-R-P-R-L-S-a-K-G-P-nle-a-f-OH  (SEQ ID NO: 75)pE-R-P-R-L-nva-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 76)pE-R-P-R-L-a-H-K-G-P-nva-a-f-OH  (SEQ ID NO: 77)pE-R-P-R-L-a-H-K-G-P-nle-nva-f-OH  (SEQ ID NO: 78)pE-R-P-R-L-S-f-K-G-P-nle-a-f-OH  (SEQ ID NO: 79)pE-R-P-R-L-S-h-K-G-P-nle-a-f-OH  (SEQ ID NO: 80)pE-R-P-R-L-a-H-K-G-P-f-a-f-OH  (SEQ ID NO: 81)pE-R-P-R-L-A-h-K-G-P-nle-a-f-OH  (SEQ ID NO: 82)pE-R-P-R-L-a-H-Q-G-P-nle-a-f-OH  (SEQ ID NO: 83)pE-R-P-R-L-a-H-E-G-P-nle-a-f-OH  (SEQ ID NO: 84)pE-R-P-R-L-v-H-K-G-P-nle-a-f-OH  (SEQ ID NO: 85)pE-R-P-R-L-a-H-K-D-P-nle-a-f-OH;  (SEQ ID NO: 86)pE-R-P-R-Cha-nva-H-K-G-P-nle-a-f-OH 

or an amide, an ester or a salt of the polypeptide. 23-25. (canceled)26. The bioconjugate or a multimer thereof, according to claim 1,wherein the half-life extending moiety is a human Serum Albumin.
 27. Thebioconjugate according to claim 26 wherein the Human Serum Albumin ischemically linked to the N-terminus of the polypeptide via a linker ofthe following Formulae:

wherein x is 1-20, R is linear or branched alkylene, cycloalkyl, aryl ofheteroaryl or combination thereof, R′ is linear or branched alkylene,aryl or cycloalkyl or combination thereof.
 28. The bioconjugateaccording to claim 26 wherein the Human Serum Albumin is chemicallylinked to the C-terminus of the polypeptide via a linker of thefollowing Formulae:

wherein x is 1-20, R is linear or branched alkylene, cycloalkyl, aryl ofheteroaryl or combination thereof, R′ is linear or branched alkylene,aryl or cycloalkyl or combination thereof.
 29. A method of treating orpreventing a disease or disorder responsive to the agonism of the APJreceptor, in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a bioconjugate accordingto claim
 1. 30. The method of claim 29 wherein the disease or disorderis selected from acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.31-33. (canceled)
 34. A combination comprising a therapeuticallyeffective amount of a bioconjugate according to claim 1, and one or moretherapeutically active co-agent.
 35. A combination according to claim 34wherein the co-agent is selected from inotropes, beta adrenergicreceptor blockers, HMG-Co-A reductase inhibitors, angiotensin IIreceptor antagonists, angiotensin converting enzyme (ACE) Inhibitors,calcium channel blockers (CCB), endothelin antagonists, renininhibitors, diuretics, ApoA-I mimics, anti-diabetic agents,obesity-reducing agents, aldosterone receptor blockers, endothelinreceptor blockers, aldosterone synthase inhibitors (ASI), a CETPinhibitor, anti-coagulants, relaxin, BNP (nesiritide) and/or a NEPinhibitor.
 36. A pharmaceutical composition comprising a therapeuticallyeffective amount of a bioconjugate according to claim 1, and one or morepharmaceutically acceptable carriers.